Proteins encoded by polynucleic acids of porcine reproductive and respiratory syndrome virus (PRRSV)

ABSTRACT

The present invention provides a purified preparation containing, for example, at least one polypeptide selected from the group consisting of proteins encoded by one or more open reading frames (ORF&#39;s) of an Iowa strain of porcine reproductive and respiratory syndrome virus (PRRSV), antigenic regions of such proteins which are at least 5 amino acids in length and which effectively protect a porcine host against a subsequent challenge with a PRRSV isolate, and combinations thereof in which amino acids non-essential for antigenicity may be conservatively substituted. The present invention also concerns a vaccine comprising an effective amount of such a protein; antibodies which specifically bind to such a protein; methods of producing the same; and methods of protecting a pig against a PRRSV, treating a pig infected by a PRRSV, and detecting PRRSV in a pig.

[0001] This application is a divisional of U.S. application Ser. No.09/019,793, filed Feb. 6, 1998, which is a continuation-in-part ofapplication Ser. No. 08/478,316, filed Jun. 7, 1995, now pending, whichis a continuation-in-part of application Seri. No. 08/301,435, filed onSep. 1, 1994, now pending, which is a continuation-in-part ofapplication Ser. No. 08/131,625, filed on Oct. 5, 1993, now pending,which is a continuation-in-part of application Ser. No. 07/969,071,filed on Oct. 30, 1992, now abandoned. The entire contents ofapplication Ser. Nos. 08/131,625, 08/301,435 and 09/019,793, filed onOct. 5, 1993, Sep. 1, 1994 and Feb. 6, 1998, respectively, areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention concerns polynucleic acids isolated from aporcine reproductive and respiratory syndrome virus (PRRSV), a proteinand/or a polypeptide encoded by the polynucleic acids, a vaccine whichprotects pigs from a PRRSV based on the protein or polynucleic acids,methods of making the proteins, polypeptides and polynucleic acids, amethod of protecting a pig from PRRS using the vaccine, a method ofproducing the vaccine, a method of treating a pig infected by or exposedto a PRRSV, and a method of detecting a PRRSV.

[0004] 2. Discussion of the Background:

[0005] Porcine reproductive and respiratory syndrome (PRRS), a new andsevere disease in swine, was first reported in the U.S.A. in 1987, andwas rapidly recognized in many western European countries (reviewed byGoyal, J. Vet. Diagn. Invest., 1993, 5:656-664; and in U.S. applicationSer. Nos. 08/131,625 and 08/301,435). The disease is characterized byreproductive failure in sows and gilts, pneumonia in young growing pigs,and an increase in preweaning mortality (Wensvoort et al., Vet. Q.,13:121-130, 1991; Christianson et al., 1992, Am. J. Vet. Res.53:485-488; U.S. Application Serial Nos. 08/131,625 and 08/301,435).

[0006] The causative agent of PRRS, porcine reproductive and respiratorysyndrome virus (PRRSV), was identified first in Europe and then in theU.S.A. (Collins et al., 1992, J. Vet. Diagn. Invest., 4:117-126). TheEuropean strain of PRRSV, designated as Lelystad virus (LV), has beencloned and sequenced (Meulenberg et al., 1993, Virology, 192:62-72 andJ. Gen. Virol., 74:1697-1701; Conzelmann et al., 1993, Virology,193:329-339).

[0007] PRRSV was provisionally classified in the proposed new virusfamily of Arteriviridae, which includes equine arteritis virus (EAV),lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fevervirus (SHFV) (Plagemann and Moennig, 1992, Adv. Virus. Res., 41:99-192;Godeny et al., 1993, Virology, 194:585-596; U.S. application Ser. Nos.08/131,625 and 08/301,435). This group of single plus-strand RNA virusesshares many characteristics such as genome organization, replicationstrategy, morphology and macrophage tropism (Meulenberg et al., 1993;U.S. application Ser. Nos. 08/131,625 and 08/301,435). Subclinicalinfections and persistent viremia with concurrent antibody productionare also characteristic histopathologic properties of the arteriviruses.

[0008] Antigenic, genetic and pathogenic variations have been reportedamong PRRSV isolates (Wensvoort et al., 1992, J. Vet. Diagn. Invest.,4:134-138; Mardassi et al., 1994, J. Gen. Virol., 75:681-685; U.S.aapplication Ser. Nos. 08/131,625 and 08/301,435). Furthermore, U.S. andEuropean PRRSV represent two distinct genotypes (U.S. application Ser.Nos. 08/131,625 and 08/301,435). Antigenic variability also exists amongdifferent North American isolates as well (Wensvoort et al., 1992).Marked differences in pathogenicity have been demonstrated not onlybetween U.S. and European isolates, but also among different U.S.isolates (U.S. application Ser. Nos. 08/131,625 and 08/301,435).

[0009] The genomic organization of arteriviruses resembles coronavirusesand toroviruses in that their replication involves the formation of a3′-coterminal nested set of subgenomic mRNAs (sg mRNAs) (Chen et al.,1993, J. Gen. Virol. 74:643-660; Den Boon et al., 1990, J. Virol.,65:2910-2920; De Vries et al., 1990, Nucleic Acids Res., 18:3241-3247;Kuo et al., 1991, J. Virol., 65:5118-5123; Kuo et al., 1992; U.S.application Ser. Nos. 08/131,625 and 08/301,435). Partial sequences ofseveral North American isolates have also been determined (U.S.application Ser. Nos. 08/131,625 and 08/301,435; Mardassi et al., 1994,J. Gen. Virol., 75:681-685).

[0010] The genome of PRRSV is polyadenylated, about 15 kb in length andcontains eight open reading frames (ORFS; Meulenberg et al., 1993; U.S.application Ser. Nos. 08/131,625 and 08/301,435). ORFs 1a and 1bprobably encode viral RNA polymerase (Meulenberg et al., 1993). ORFs 5,6 and 7 were found to encode a glycosylated membrane protein (E), anunglycosylated membrane protein (M) and a nucleocapsid protein (N),respectively (Meulenberg et al., 1995). ORFs 2 to 4 appear to have thecharacteristics of membrane-associated proteins (Meulenberg et al.,1993; U.S. application Ser. No. 08/301,435). However, the translationproducts of ORFs 2 to 4 were not detected in virus-infected cell lysatesor virions (Meulenberg et al., 1995).

[0011] The major envelope glycoprotein of EAV encoded by ORF 5 may bethe virus attachment protein, and neutralizing monoclonal antibodies(MAbs) are directed to this protein (de Vries, J. Virol., 1992;66:6294-6303; Faaberg, J. Virol., 1995; 69:613-617). The primaryenvelope glycoprotein of LDV, a closely related member of PRRSV, is alsoencoded by ORF 5, and several different neutralizing MAbs were found tospecifically immunoprecipitate the ORF 5 protein (Cafruny et al., Vir.Res., 1986; 5:357-375). Therefore, it is likely that the major envelopeprotein of PRRSV encoded by ORF 5 may induce neutralizing antibodiesagainst PRRSV.

[0012] It has been proposed that antigenic variation of viruses is theresult of direct selection of variants by the host immune responses(reviewed by Domingo et al., J. Gen. Virol., 1993, 74:2039-2045). Thus,these hypervariable regions are likely due to the host immune selectionpressure and may explain the observed antigenic diversity among PRRSVisolates.

[0013] The M and N proteins of U.S. PRRSV isolates, including ISU 3927,are highly conserved (U.S. application Ser. No. 08/301,435). The M and Nproteins are integral to preserving the structure of PRRSV virions, andthe N protein may be under strict functional constraints. Therefore, itis unlikely either that (a) the M and N proteins are subjected to majorantibody selection pressure or that (b) ORFs 6 and 7, which are likelyto encode the M and N proteins, are responsible for or correlated toviral virulence. Interestingly, however, higher sequence variation ofthe LDV M protein was observed between LDV isolates with differingneurovirulence (Kuo et al., 1992, Vir. Res. 23:55-72).

[0014] ORFs la and lb are predicted to translate into a single protein(viral polymerase) by frameshifting. ORFs 2 to 6 may encode the viralmembrane associated proteins.

[0015] In addition to the genomic RNA, many animal viruses produce oneor more sg mRNA species to allow expression of viral genes in aregulated fashion. In cells infected with PRRSV, seven species ofvirus-specific mRNAs representing a 3′-coterminal nested set aresynthesized (mRNAs 1 to 7, in decreasing order of size). mRNA 1represents the genomic mRNA. Each of the sg mRNAs contains a leadersequence derived from the 5′-end of the viral genome.

[0016] The numbers of the sg mRNAs differ among arteriviruses and evenamong different isolates of the same virus. A nested set of 6 sg mRNAswas detected in EAV-infected cells and European PRRSV-infected cells.However, a nested set of six (LDV-C) or seven (LDV-P) sg mRNAs, inaddition to the genomic RNA, is present in LDV-infected cells. Theadditional sg mRNA 1-1 of LDV-P contains the 3′-end of ORF 1b and canpotentially be translated to a protein which represents the C-terminalend of the viral polymerase. Sequence analysis of the sg mRNAs of LDVand EAV indicates that the leader-mRNA junction motif is conserved.Recently, the leader-mRNA junction sequences of the European LV werealso shown to contain a common motif, UCAACC, or a highly similarsequence.

[0017] The sg mRNAs have been shown to be packaged into the virions insome coronaviruses, such as bovine coronavirus (BCV) and transmissiblegastroenteritis virus (TGEV). However, only trace amounts of the sgmRNAs were detected in purified virions of mouse hepatitis virus (MHV),another coronavirus. The sg mRNAs of LDV, a closely related member ofPRRSV, are also not packaged in the virions, and only the genomic RNAwas detected in purified LDV virions.

[0018] The sg mRNAs of LDV and EAV have been characterized in detail.However, information regarding the sg mRNAs of PRRSV strains, especiallythe U.S. PRRSV, is very limited. Thus, a need is felt for a morethorough molecular characterization of the sg mRNAs of U.S. PRRSV.

[0019] The packaging signal of MHV is located in the 3′-end of ORF lb,thus only the genomic RNA of MHV is packaged. The sg mRNAs of BCV andTGEV, however, are found in purified virions. The packaging signal ofBCV and TGEV has not been determined. The Aura alphavirus sg mRNA isefficiently packaged into the virions, presumably because the packagingsignal is present in the sg mRNA. The Sindbis virus 26S sg mRNA is notpackaged into virions because the packaging signal is located in thegenome segment (not present in sg mRNA). The sg mRNAs of LDV, a closelyrelated member of PRRSV, are also not packaged into the virions.

[0020] Many mechanisms are involved in the generation of the sg mRNAs.It has been proposed that coronaviruses utilize a unique leaderRNA-primed transcription mechanism in which a leader RNA is transcribedfrom the 3′ end of the genome-sized negative-stranded template RNA,dissociates from the template, and then rejoins the template RNA atdownstream intergenic regions to prime the transcription of sg mRNAs.The model predicts that the 5 ′-leader contains a specific sequence atits 3′-end which is repeated further downstream in the genome, precedingeach of the ORFs 2 to 7. The leader joins to the body of each of the sgmRNAs via the leader-mRNA junction segment.

[0021] PRRSV is an important cause of pneumonia in nursery and weanedpigs. PRRSV causes significant economic losses from pneumonia in nurserypigs (the exact extent of which are not fully known). Reproductivedisease was the predominant clinical outcome of PRRSV infections duringthe past few years, due to the early prevalence of relatively lowvirulence strains of PRRSV. Respiratory disease has now become the mainproblem associated with PRRSV, due to the increasing prevalence ofrelatively high virulence strains of PRRSV. A need is felt for a vaccineto protect against disease caused by the various strains of PRRSV.

[0022] Surprisingly, the market for animal vaccines in the U.S. andworldwide is larger than the market for human vaccines. Thus, thereexists an economic incentive to develop new veterinary vaccines, inaddition to the substantial public health benefit which is derived fromprotecting farm animals from disease.

SUMMARY OF THE INVENTION

[0023] Accordingly, one object of the present invention is to provide apolynucleic acid isolated from a porcine reproductive and respiratorysyndrome virus (PRRSV).

[0024] It is a further object of the present invention to provide anisolated polynucleic acid which encodes a PRRSV protein.

[0025] It is a further object of the present invention to provide aPRRSV protein, either isolated from a PRRSV or encoded by a PRRSVpolynucleic acid.

[0026] It is a further object of the present invention to provide aprotein- or polynucleic acid-based vaccine which protects a pig againstPRRS.

[0027] It is a further object of the present invention to provide amethod of raising an effective immunological response against a PRRSVusing the vaccine.

[0028] It is a further object of the present invention to provide amethod of producing a protein- or polynucleic acid-based vaccine whichprotects a pig against PRRS.

[0029] It is a further object of the present invention to provide amethod of treating a pig exposed to a PRRSV or suffering from PRRS.

[0030] It is a further object of the present invention to provide amethod of detecting PRRSV.

[0031] It is a further object of the present invention to provide anantibody which immunologically binds to a PRRSV protein or to anantigenic region of such a protein.

[0032] It is a further object of the present invention to provide anantibody which immunologically binds to a protein- or polynucleicacid-based vaccine which protects a pig against a PRRSV.

[0033] It is a further object of the present invention to provide adiagnostic kit for assaying or detecting a PRRSV.

[0034] It is a further object of the present invention to provide theabove objects, where the PRRS virus is an Iowa strain of PRRSV.

[0035] These and other objects, which will become apparent during thefollowing description of the preferred embodiments, have been providedby a purified and/or isolated polypeptide selected from the groupconsisting of proteins encoded by one or more open reading frames(ORF's) of an Iowa strain of porcine reproductive and respiratorysyndrome virus (PRRSV), proteins at least 94% but less than 100%homologous with a protein encoded by an ORF 2 of an Iowa strain ofPRRSV, proteins at least 88% but less than 100% homologous with aprotein encoded by ORF 3 of an Iowa strain of PRRSV, proteins at least93% homologous with an ORF 4 of an Iowa strain of PRRSV, proteins atleast 90% homologous with an ORF 5 of an Iowa strain of PRRSV, proteinsat least 97% but less than 100% homologous with proteins encoded by oneor both of ORF 6 and ORF 7 of an Iowa strain of PRRSV, antigenic regionsof such proteins which are at least 5 amino acids in length and whicheffectively stimulate protection in a porcine host against a subsequentchallenge with a PRRSV isolate, and combinations thereof; an isolatedpolynucleic acid which encodes such a polypeptide or polypeptides; avaccine comprising an effective amount of such a polynucleotide orpolypeptide(s); antibodies which specifically bind to such apolynucleotide or polypeptide; methods of producing the same; andmethods of (i) effectively protecting a pig against PRRS, (ii) treatinga pig exposed to a PRRSV or suffering from PRRS, and (iii) detecting aPRRSV using the same.

BRIEF DESCRIPTION OF THE FIGURES

[0036]FIG. 1A, 1B, 1C, 1D, 1E, 1F and 1G shows a nucleotide sequencecomparison of ORFs 2 to 5 of U.S. isolates ISU 79 (SEQ ID NO:7), ISU1894, ISU 3927, ISU 22 (SEQ ID NO:4) and ISU 55 (SEQ ID NO:3) with otherknown PRRSV isolates (VR 2385: SEQ ID NO: 1, VR 2332: SEQ ID NO:5);

[0037]FIGS. 2A, 2B, 2C and 2D respectively show the alignment of thededuced amino acid sequences of ORF 2, ORF 3, ORF 4 and ORF 5 of U.S.isolates ISU 79 (SEQ ID NOS: 10, 18, 29, 36, respectively), ISU 1894(SEQ ID NOS: 12, 19, 27 and 35, respectively), ISU 22 (SEQ ID NOS: 9,20, 28 and 37, respectively), ISU 55 (SEQ ID NOS: 11, 17, 26 and 34,respectively) and ISU 3927 (SEQ ID NOS: 13, 21, 20 and 38, respectively)with other known PRRSV isolates (VR 2385: SEQ ID NOS: 8, 15, 24 and 32,respectively; VR 2332: SEQ ID NOS: 14, 22, 25 and 33, respectively; LV:SEQ ID NOS: 15, 23, 31 and 39, respectively);

[0038]FIG. 3 shows a phylogenetic tree based on the nucleotide sequencesof ORFs 2 to 7 of seven U.S. PRRSV isolates with differing virulence;

[0039]FIG. 4 shows a Northern blot analysis of RNAs isolated from ISU3927-infected CRL 11171 cells (lane 1) and from purified virions of ISU3927 (lane 2);

[0040]FIG. 5 shows a Northern blot analysis of total intracellular RNAsisolated from CRL 11171 cells infected with ISU22 (lane 1), ISU 55 (lane2), ISU 79 (lane 3), ISU 1894 (lane 4) and ISU 3927 (lane 5),respectively;

[0041]FIGS. 6A and 6B show a Northern hybridization of total RNAsisolated from CRL 11171 cells infected with ISU 79 at differentmultiplicities of infection (m.o.i.) (A), and polyadenylated RNA fromcells-infected with PRRSV isolates ISU 55 and ISU 79 (B);

[0042]FIGS. 7A and 7B show a Northern blot analysis of totalintracellular mRNAs isolated from CRL 11171 cells infected with ISU 1894(A) and ISU 79 (B);

[0043]FIGS. 8A and 8B show RT-PCR amplification of the 5′-terminalsequences of the sg mRNAs 3 and 4 of ISU 1894 (lane 1) and sg mRNAs 3, 4and 4-1 of ISU 79 (lane 2) (A) where lane L is a 1-kb marker; and theleader-mRNA junction sequences of sg mRNAs 3 and 4 of ISU 79 and ISU1894 and of sg MRNA 4-1 of ISU 79 (B), where the locations of theleader-mRNA junction sequences in the genomes relative to the startcodon of each ORF were indicated by minus (−) numbers of nucleotidesupstream of the ORFS; and

[0044]FIG. 9A, 9B, 9C and 9D shows the sequence alignment of ORFs 2 to 7of ISU 1894 (SEQ ID NO:41) and ISU 79 (SEQ ID NO:40), where the startcodon of each ORF is indicated by +>, the termination codon of each ORFis indicated by asterisks (*), the determined or predicted leader-mRNAjunction sequences are underlined and the locations of the leader-mRNAjunction sequences relative to the start codon of each ORF are indicatedby minus (−) numbers of nucleotides upstream of each ORF.

[0045]FIG. 10. Immunofluorescence assay of the MAbs with PRRSV-infectedcells. Hybridoma supernatant was tested with IFA on infected ATCC CRL11171 cells. Typical immunofluorescence from reaction withprotein-specific MAbs is shown here. A. GP4-specific MAb, PP4bB3; B.E-specific MAb, PP5dB4; C. N-specific MAb, PP7eFl 1; and D. Negativecontrol, PPAc8.

[0046]FIG. 11. Reactivity of the MAbs and detergent extracted PRRSVantigen in ELISA. Plates were coated with antigen extracted fromPRRSV-infected cells with detergent 1% Triton X-100 and blocked with 1%BSA. Hybridoma supernatant was tested along with positive and negativecontrols, PP7eFl 1 and PPACS respectively. Specific reactions weredetected with anti-mouse IgG peroxidase conjugate. ABTS substrate wasincubated in the plates for 20 min before A405 was measured. The firstfour MAbs starting from PP4bB3 are GP4-specific antibodies, and the nextsix MAbs starting from PP5bH4 are E-specific antibodies.

[0047]FIG. 12. Reactivity of the E specific MAbs and extract of PRRSVvirions in Immunoblotting. MW standards (in kDa) are indicated on theleft side of the figure. Lanes: 1, PP5dB4; 2, PP5bH4; 3, Negativecontrol: PPAc8; 4, Positive control: pig anti-PRRSV serum; 5, Negativecontrol: normal pig serum.

[0048]FIG. 13. Titers of monoclonal antibodies.

[0049]FIG. 14. Reactivity pattern of PRRSSV isolates with the MAbs toPRRSV. Titers of the MAbs were shown in FIG. 13. The reactivity patternwas determined according to the titers of at least 6 MAbs with any oneisolate: <=32—low reactivity; 64 to 128—medium reactivity; >=256—highreactivity. Those isolates not belonging to the groups above weregrouped as other. Total isolates tested were 23.

[0050]FIG. 15. Primers used to amplify PRRSV ORFs 2 through 7 genes withPCR.

[0051]FIG. 16. Recombinant proteins of PRRSV ORFs 2 to 5 expressed ininsect cells. a=predicted M_(r) of products of PRRSV ORFs 2 to 5 andN-glycosylation sites are based on nucleotide sequence studies (Meng etal, 1994 & Morozov et al, 1995). b=expressed products in insect cells.c=bands after tunicamycin treatment were determined by immunoblottinganalysis. d=leader-free core proteins are determined on the basis oftunicamycin treatment analysis. The presence of the other bands in therecombinant products after tunicamycin treatment was possibly due toO-linked glycosylation, phosphorylation or other post-translationalmodifications.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] In the present application, the nucleotide sequences of the ORFs2 to 5 of a low virulence isolate and four other Iowa strain PRRSVisolates with “moderate” and high virulence have been determined. Basedon comparisons of ORFs 2 to 7 of various PRRSV isolates, the leastvirulent U.S. isolate known (ISU 3927) has relatively high sequencevariations in ORFs 2 to 4, as compared to the variations in other U.S.isolates. Furthermore, based on analysis of the sequences of the ORFS,at least three minor genotypes exist within the major genotype of U.S.PRRSV.

[0053] Sequence analysis of the ORF 5 protein of different PRRSVisolates reveals three hypervariable regions which containednon-conserved amino acid substitutions. These regions are hydrophilicand also antigenic as predicted by computer analysis.

[0054] In the present invention, a “porcine reproductive and respiratorysyndrome virus” or “PRRSV” refers to a virus which causes the diseasesPRRS, PEARS, SIRS, MSD and/or PIP (the term “PIP” now appears to bedisfavored), including the Iowa strain of PRRSV, other strains of PRRSVfound in the United States (e.g., VR 2332), strains of PRRSV found inCanada (e.g., IAF-exp91), strains of PRRSV found in Europe (e.g.,Lelystad virus, PRRSV-10), and closely-related variants of these viruseswhich may have appeared and which will appear in the future.

[0055] The “Iowa strain” of PRRSV includes (a) PRRSV isolates depositedin the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2205, USA, by the present inventors and/or describedin this application and/or in either of prior U.S. application Ser. Nos.08/131,625 and 08/301,435, (b) PRRS viruses which produce more than sixsg mRNAs when cultured or passaged in CRL 11171 cells, (c) PRRSVs whichproduce at least 40% gross lung lesions or lung consolidation in5-week-old caesarean-derived, colostrum-deprived piglets 10 dayspost-infection, (d) a PRRSV isolate having a genome which encodes aprotein having the minimum homology to a PRRSV ORF described in Table 2below, and/or (d) any PRRSV isolate having the identifyingcharacteristics of such a virus.

[0056] The present vaccine is effective if it protects a pig againstinfection by a porcine reproductive and respiratory syndrome virus(PRRSV). A vaccine protects a pig against infection by a PRRSV if, afteradministration of the vaccine to one or more unaffected pigs, asubsequent challenge with a biologically pure virus isolate (e.g., VR2385, VR 2386, or other virus isolate described below) results in alessened severity of any gross or histopathological changes (e.g.,lesions in the lung) and/or of symptoms of the disease, as compared tothose changes or symptoms typically caused by the isolate in similarpigs which are unprotected (i.e., relative to an appropriate control).More particularly, the present vaccine may be shown to be effective byadministering the vaccine to one or more suitable pigs in need thereof,then after an appropriate length of time (e.g., 1-4 weeks), challengingwith a large sample (10³⁻⁷ TCID₅₀) of a biologically pure PRRSV isolate.A blood sample is then drawn from the challenged pig after about oneweek, and an attempt to isolate the virus from the blood sample is thenperformed (e.g., see the virus isolation procedure exemplified inExperiment VIII below). Isolation of the virus is an indication that thevaccine may not be effective, and failure to isolate the virus is anindication that the vaccine may be effective.

[0057] Thus, the effectiveness of the present vaccine may be evaluatedquantitatively (i.e., a decrease in the percentage of consolidated lungtissue as compared to an appropriate control group) or qualitatively(e.g., isolation of PRRSV from blood, detection of PRRSV antigen in alung, tonsil or lymph node tissue sample by an immunoperoxidase assaymethod [described below], etc.). The symptoms of the porcinereproductive and respiratory disease may be evaluated quantitatively(e.g., temperature/fever), semi-quantitatively (e.g., severity ofrespiratory distress [explained in detail below]), or qualitatively(e.g., the presence or absence of one or more symptoms or a reduction inseverity of one or more symptoms, such as cyanosis, pneumonia, heartand/or brain lesions, etc.).

[0058] An unaffected pig is a pig which has either not been exposed to aporcine reproductive and respiratory disease infectious agent, or whichhas been exposed to a porcine reproductive and respiratory diseaseinfectious agent but is not showing symptoms of the disease. An affectedpig is one which shows symptoms of PRRS or from which PRRSV can beisolated.

[0059] The clinical signs or symptoms of PRRS may include lethargy,respiratory distress, “thumping” (forced expiration), fevers, roughenedhaircoats, sneezing, coughing, eye edema and occasionallyconjunctivitis. Lesions may include gross and/or microscopic lunglesions, myocarditis, lymphadenitis, encephalitis and rhinitis. Inaddition, less virulent and non-virulent forms of PRRSV and of the Iowastrain have been found, which may cause either a subset of the abovesymptoms or no symptoms at all. Less virulent and non-virulent forms ofPRRSV can be used according to the present invention to provideprotection against porcine reproductive and respiratory diseasesnonetheless.

[0060] The phrase “polynucleic acid” refers to RNA or DNA, as well asmRNA and cDNA corresponding to or complementary to the RNA or DNAisolated from the virus or infectious agent. An “ORF” refers to an openreading frame, or polypeptide-encoding segment, isolated from a viralgenome, including a PRRSV genome. In the present polynucleic acid, anORF can be included in part (as a fragment) or in whole, and can overlapwith the 5′- or 3′-sequence of an adjacent ORF (see for example, FIG. 1and Experiment 1 below). A “polynucleotide” is equivalent to apolynucleic acid, but may define a distinct molecule or group ofmolecules (e.g., as a subset of a group of polynucleic acids).

[0061] In the Experiments described hereinbelow, the isolation, cloningand sequencing of ORFs 2 to 5 of (a) a low virulence U.S. PRRSV isolateand (b) two other U.S. PRRSV isolates of varying virulence weredetermined. The nucleotide and deduced amino acid sequences of thesethree U.S. isolates were compared with the corresponding sequences ofother known PRRSV isolates (see, for example, U.S. application Ser. No.08/301,435). The results indicate that considerable genetic variationsexist not only between U.S. PRRSV and European PRRSV, but also among theU.S. isolates as well.

[0062] The amino acid sequence identity between the seven U.S. PRRSVisolates studied was 91-99% in ORF 2, 86-98% in ORF 3, 92-99% in ORF 4and 88-97% in ORF 5. The least virulent U.S. isolate known (ISU 3927)has higher sequence variations in ORFs 2 to 4 than in ORFs 5 to 7, ascompared to other U.S. isolates. Three hypervariable regions withantigenic potential have been identified in the major envelopeglycoprotein encoded by ORF 5.

[0063] Pairwise comparison of the sequences of ORFs 2 to 7 andphylogenetic tree analysis implied the existence of at least threegroups of PRRSV variants (or minor genotypes) within the major genotypeof U.S. PRRSV. The least virulent U.S. isolate known forms a distinctbranch from other U.S. isolates with differing virulence. The results ofthis study have implications for the taxonomy of PRRSV and vaccinedevelopment.

[0064] In a further experiment, the sg mRNAs in PRRSV-infected cellswere characterized. The data showed that a 3′-coterminal nested set ofsix or seven sg mRNAs is formed in cells infected with differentisolates of PRRSV. However, unlike some of the coronaviruses andalphavirus, the sg mRNAs of PRRSV are not packaged into the virion, andonly the genomic RNA of PRRSV was detected in purified virions.Variations in the numbers of the sg mRNAs among different PRRSV isolateswith differing virulence were also observed. Further sequence analysisof ORFs 2 to 7 of two U.S. isolates and their comparison with theEuropean LV reveal the heterogeneic nature of the leader-mRNA junctionsequences of PRRSV.

[0065] As demonstrated in Experiment 2 below, a 3′-coterminal nested setof six or more sg mRNAs is formed in cells infected with differentisolates of PRRSV. The presence of a nested set of sg mRNAs furtherindicates that U.S. PRRSV, like the European isolate Lelystad virus(LV), belongs to the newly proposed Arteriviridae family-including LDV,EAV and SHFV. Northern blot analysis with ORF-specific probes indicatesthat the structure of the PRRSV sg mRNAs is polycistronic, and each ofthe sg mRNAs except for sg mRNA 7 contains multiple ORFS. Therefore, thesequence of each sg mRNA is contained within the 3′-portion of the nextlarger sg MRNA, and not all 5′-ends of the sg mRNAs overlap with thesequences of the smaller sg mRNAs.

[0066] There is no apparent correlation, however, between the numbers ofsg mRNAs and viral pneumovirulence. An additional species, sg mRNA 4-1,was found to contain a small ORF (ORF 4-1) with a coding capacity of 45amino acids at its 5′-end.

[0067] In Experiment 2 below, the sg mRNAs of PRRSV are shown not to bepackaged into the virions. Whether sg mRNAs are packaged into virionsmay depend an whether the sg mRNAs contain a packaging signal. Since thesg mRNAs of PRRSV are not packaged into virions, the encapsulationsignal of PRRSV is likely localized in the ORF 1 region which is uniqueto the viral genome, but which is not present in the sg mRNAs.

[0068] In Experiment 2 below, the junction segments (the leader-mRNAjunction sequences) of sg mRNAs 3 and 4 of two U.S. isolates of PRRSV,ISU 79 and ISU 1894, are determined. The knowledge of the leader-mRNAjunction sequence identities provides means for effectively producing(a) chimeric viruses to be used as an infectious clone and/or as avaccine, and (b) vectors for inserting or “shuttling” one or more genesinto a suitable, infectable host. Methods for designing and producingsuch chimeric viruses, infectious clones and vectors are known (see, forexample, Sambrook et al, “Molecular Cloning: A Laboratory Manual”, 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

[0069] The leader-mRNA junction sequence of sg mRNAs 3 and 4 of the twoisolates are different (TTGACC for mRNA 4-1 of ISU 79, GTAACC for mRNA3, and TTCACC for mRNA 4). Most of the nucleotide differences in thejunctions are present in the first 3 nucleotides. The last 3 nucleotidesare invariable, suggesting that the joining of the leader sequence tothe bodies of sg mRNAs occurs within the 5′-end of the leader-mRNAjunction sequence. Similar observations have been reported for LV, EAVand LDV.

[0070] The acquisition of the additional sg mRNA 4-1 in isolate ISU 79is due to a single nucleotide substitution which generates a new leadermRNA junction sequence. This substitution occurs in the last nucleotideof the junction segment, suggesting that the last nucleotide of theleader-mRNA junction motif is critical for the binding of the leader andfor the initiation of transcription.

[0071] Although the sequence homology between the leader and theintergenic regions of coronaviruses led to the hypothesis thatbasepairing might be essential in the leader-primed transcription, noexperimental evidence has documented for the requirement of base-pairingin transcription of the sg mRNAs. For example, the sequence at the3′-end of the leader of both coronaviruses and arteriviruses that isinvolved in the fusion process remains unknown.

[0072] Several lines of evidence support the leader-primed transcriptionmechanism for coronaviruses, but the presence of negative-stranded sgmRNAs and sg replicative intermediates (sg RI) in coronavirus-infectedcells suggests that the mechanism involved in sg mRNA synthesis is morecomplex than mere base-pairing of the leader sequence with a junctionsequence. However, negative-stranded sg mRNAs have not been detected inarteriviruses except for LDV, and sg RIs have been detected only inEAV-infected cells. Therefore, sg mRNA synthesis in arteriviruses, andparticularly in PRRSV, may be less complicated than in coronaviruses.

[0073] Sequence analysis of the ORFs 2 to 7 of two U.S. PRRSV isolatesand comparison of the sequences with LV reveals the heterogeneity of theleader-mRNA junction sequences. The presence of the leader-mRNA junctionmotifs at positions which do not correspond to a sg mRNA raises aquestion as to whether the short stretch of only six nucleotides whichare conserved in the leader and junction sequences in the genomes ofPRRSV and other arteriviruses is sufficient for efficient binding of theleader to these specific junction sites upstream of the ORFS. Thisapparent discrepancy, however, may be explained by the following twopossibilities.

[0074] First, additional structural elements, such as secondarystructures or the sequences surrounding the leader-mRNA junctionsegment, are expected to be involved in the fusion (binding) of theleader to the specific sites. It has been shown that, in MHV, thesequence flanking the consensus sequence (leader-mRNA junction sequence)of UCUAAAC affects the efficiency of sg DI RNA transcription, and thatthe consensus sequence was necessary but not sufficient in and of itselffor the synthesis of the DI mRNA.

[0075] Second, the distance between two leader-mRNA junction regions mayaffect the transcription of sg mRNAs. It has been demonstrated that thedownstream leader-mRNA junction region was suppressing sg DI RNAsynthesis of MHV from the upstream leader-mRNA junction region. Thesuppression was significant when the two leader-mRNA junction sequenceseparation was less than 35 nucleotides. However, significant inhibitionof larger sg DI RNA synthesis (from the upstream leader-mRNA junctionsequence) was not observed when the two leader-mRNA junction regionswere separated by more than 100 nucleotides.

[0076] The previously reported experimental results are consistent withthe observations reported in Experiment 2 below, where an additionalspecies of sg RNA 4-1, in addition to the sg mRNA 4, is observed in someof the PRRSV isolates. The leader-mRNA junction-sequences of sg mRNAs 4and 4-1 in the Iowa strain of PRRSV are separated by about 226nucleotides. Therefore, the synthesis of the larger sg mRNA 4-1 from theupstream leader-mRNA junction sequence is not suppressed by the presenceof the downstream leader-mRNA 4 junction sequence.

[0077] In contrast, multiple potential leader-mRNA junction sequenceswere found at different positions upstream of ORFs 3, 5, 6 and 7, butthere were no sg mRNAs corresponding to these leader-mRNA junctionmotifs in the Northern blot analysis. Most of these leader-mRNA junctionsequences are separated by less than 50 nucleotides from the downstreamleader-mRNA junction region, except for ORF 7 (in which the twopotential leader-mRNA junction sequences are separated by 114nucleotides). However, sg mRNA 7 in Northern blot analysis showed awidely-diffused band. Therefore, transcription of the larger sg mRNA 7from the upstream leader-mRNA junction sequence may not be significantlysuppressed by the downstream junction sequence, but it is not easilydistinguishable from the abundant sg mRNA 7 by Northern blot analysis.

The Present Polynucleotides and Polypeptides

[0078] ORF's 2-7 of plaque-purified PRRSV isolate ISU-12 (deposited onOct. 30, 1992, in the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2205, U.S.A., under the accession numbersVR 2385 [3× plaque-purified] and VR 2386 (non-plaque-purified]) andORF's 6-7 of PRRSV isolates ISU-22, ISU-55, ISU-3927 (deposited on Sep.29, 1993, in the American Type Culture Collection under the accessionnumbers VR 2429, VR 2430 and VR 2431, respectively), ISU-79 and ISU-1894(deposited on Aug. 31, 1994, in the American Type Culture Collectionunder the accession numbers VR 2474 and VR 2475, respectively) aredescribed in detail in U.S. application Ser. No. 08/301,435. However,the techniques used to isolate, clone and sequence these genes can bealso applied to the isolation, cloning and sequencing of the genomicpolynucleic acids of any PRRSV. Thus, the present invention is notlimited to the specific sequences disclosed in the Experiments below.

[0079] For example, primers for making relatively large amounts of DNAby the polymerase chain reaction (and if desired, for making RNA bytranscription and/or protein by translation in accordance with known invivo or in vitro methods) can be designed on the basis of sequenceinformation where more than one sequence obtained from a PRRSV genomehas been determined (e.g., ORF's 2-7 of VR 2385, VR 2429, VR 2430, VR2431, VR 2474, ISU-1894, VR 2332 and Lelystad virus). A region fromabout 15 to 50 nucleotides in length having at least 80% and preferablyat least 90% identity is selected from the determined sequences. Aregion where a deletion occurs in one of the sequences (e.g., of atleast 5 nucleotides) can be used as the basis for preparing a selectiveprimer for selective amplification of the polynucleic acid of one strainor type of PRRSV over another (e.g., for the differential diagnosis ofNorth American and European PRRSV strains).

[0080] Once the genomic polynucleic acid is amplified and cloned into asuitable host by known methods, the clones can be screened with a probedesigned on the basis of the sequence information disclosed herein. Forexample, a region of from about 50 to about 500 nucleotides in length isselected on the basis of either a high degree of identity (e.g., atleast 90%) among two or more sequences (e.g., in ORF's 6-7 of the Iowastrains of PRRSV disclosed in Experiment III below), and apolynucleotide of suitable length and sequence identity can be preparedby known methods (such as automated synthesis, or restriction of asuitable fragment from a polynucleic acid containing the selectedregion, PCR amplification using primers which hybridize specifically tothe polynucleotide, and isolation by electrophoresis). Thepolynucleotide may be labeled with, for example, ³²p (for radiometricidentification) or biotin (for detection by fluorometry). The probe isthen hybridized with the polynucleic acids of the clones and detectedaccording to known methods.

[0081] The present inventors have discovered that one or more of ORFs2-4 may be related to the virulence of PRRSV. For example, at least oneisolate of PRRSV which shows relatively low virulence also appears tohave a deletion in ORF 4 (see, for example, Experiments VIII-XI in U.S.application Ser. No. 08/301,435). Furthermore, the least virulent knownisolate (VR 2431) shows a relatively high degree of variance in bothnucleotide and amino acid sequence information in ORFs 2-4, as comparedto other U.S. PRRSV isolates. Thus, in one embodiment, the presentinvention concerns polynucleotides and polypeptides related to ORFs 2-4of VR 2431.

[0082] In a further embodiment, the present invention is concerned witha polynucleic acid obtained from a PRRSV isolate which confersimmunogenic protection directly or indirectly against a subsequentchallenge with a PRRSV, but in which the polynucleic acid is deleted ormutated to an extent which would render a PRRSV containing thepolynucleic acid either low-virulent (i.e., a “low virulence” (lv)phenotype; see the corresponding explanation in U.S. application Ser.No. 08/301,435) or non-virulent (a so-called “deletion mutant”).Preferably, one or more of ORFs 2-4 is/are deleted or mutated to anextent which would render a PRRS virus non-virulent. However, it may bedesirable to retain regions of one or more of ORFs 2-4 in the presentpolynucleic acid which (i) encode an antigenic and/or immunoprotectivepeptide fragment and which (ii) do not confer virulence to a PRRS viruscontaining the polynucleic acid.

[0083] The present invention also encompasses a PRRSV per se in whichone or more of ORFs 2-4 is deleted or mutated to an extent which rendersit either low-virulent or non-virulent (e.g., VR 2431). Such a virus isuseful as a vaccine or as a vector for transforming a suitable host(e.g., MA-104, PSP 36, CRL 11171, MARC-145 or porcine alveolarmacrophage cells) with a heterologous gene. Preferred heterologous geneswhich may be expressed using the present deletion mutant may includethose encoding a protein or an antigen other than a porcine reproductiveand respiratory syndrome virus antigen (e.g., pseudorabies and/or swineinfluenza virus proteins and/or polypeptide-containing antigens, aporcine growth hormone, etc.) or a polypeptide-based adjuvant (such asthose discussed in U.S. application Ser. No. 08/301,435 for a vaccinecomposition).

[0084] It may also be desirable in certain embodiments of the presentpolynucleic acid which contain, for example, the 3′-terminal region of aPRRSV ORF (e.g., from 200 to 700 nucleotides in length), at least partof which may overlap with the 5′-region of the ORF immediatelydownstream. Similarly, where the 3′-terminal region of an ORF mayoverlap with the 5′-terminal region of the immediate downstream ORF, itmay be desirable to retain the 5′-region of the ORF which overlaps withthe ORF immediately downstream.

[0085] The present inventors have also discovered that ORF 5 in thePRRSV genome appears to be related to replication of the virus inmammalian host cells capable of sustaining a culture while infected withPRRSV. Accordingly, the present invention is also concerned withpolynucleic acids obtained from a PRRSV genome in which ORF 5 may bepresent in multiple copies (a so-called “overproduction mutant”). Forexample, the present polynucleic acid may contain at least two, and morepreferably, from 2 to 10 copies of ORF 5 from a high-replication (hr)phenotype PRRSV isolate.

[0086] Interestingly, the PRRSV isolate ISU-12 has a surprisingly largenumber of potential start codons (ATG/AUG sequences) near the5′-terminus of ORF 5, possibly indicating alternate start sites of thisgene. Thus, alternate forms of the protein encoded by ORF 5 of a PRRSVisolate may exist, particularly where alternate ORF's encode a proteinhaving a molecular weight similar to that determined experimentally(e.g., from about 150 to about 250 amino acids in length). The mostlikely coding region for ORF 5 of ISU-12 is indicated in FIG. 1.

[0087] One can prepare deletion and overproduction mutants in accordancewith known methods. For example, one can prepare a mutant polynucleicacid which contains a “silent” or degenerate change in the sequence of aregion encoding a polypeptide. By selecting and making an appropriatedegenerate mutation, one can substitute a polynucleic acid sequencerecognized by a known restriction enzyme (see, for example, Experiment 2below). Thus, if a silent, degenerate mutation is made at one or two ofthe 3′-end of an ORF and the 5′-end of a downstream ORF, one can inserta synthetic polynucleic acid (a so-called “cassette”) which may containa polynucleic acid encoding one or multiple copies of an hr ORF 5protein product, of a PRRSV or other viral envelope protein and/or anantigenic fragment of a PRRSV protein. The “cassette” may be preceded bya suitable initiation codon (ATG), and may be suitably terminated with atermination codon at the 3′-end (TAA, TAG or TGA). Of course, anoligonucleotide sequence which does not encode a polypeptide may beinserted, or alternatively, no cassette may be inserted. By doing so,one may provide a so-called deletion mutant.

[0088] The present invention also concerns regions and positions of thepolypeptides encoded by ORFs of VR 2431 which may be responsible for thelow virulence of this isolate. Accordingly, the present isolated and/orpurified polypeptide may be one or more encoded by a “low-virulencemutation” of one or more of ORFs 2, 3 and 4 of a PRRSV (or alow-virulence fragment thereof at least 5 amino acids in length) inwhich one or more of positions 12-14 of the polypeptide encoded by ORF 2are RGV (in which “R”, “G” and “V” are the one-letter abbreviations forthe corresponding amino acids), positions 44-46 are LPA, position 88 isA, position 92 is R, position 141 is G, position 183 is H, position 218is S, position 240 is S and positions 252-256 are PSSSW (SEQ ID NO:42),or any combination thereof. Other amino acid residue identities whichcan be further combined with one or more of the above amino acidposition identities include those at position 174 (I) and position 235(M).

[0089] The present isolated and/or purified polypeptide may also be oneencoded by an ORF 3 of a PRRSV in which one or more of the specifiedamino acid identities may be selected from those at positions 11 (L), 23(V), 26-28 (TDA), 65-66 (QI), 70 (N), 79 (N), 93 (T), 100-102 (KEV), 134(K), 140 (N), 223-227 (RQRIS; SEQ ID NO:43), 234 (A) and 235 (M), or anycombination thereof, which may be further combined with one or more ofpositions 32 (F), 38 (M), 96 (P), 143 (L), 213-217 (FQTS; SEQ ID NO:44),231 (R), and 252 (A).

[0090] The present isolated and/or purified polypeptide may also be oneencoded by an ORF 4 of a PRRSV in which one or more of the specifiedamino acid identities may be selected from those at positions 13 (E), 43(N), 56 (G), 58-59 (TT), 134 (T), 139 (I) and any combination thereof,which may be further combined with one or more of positions 2-3 (AA), 51(G) and 63 (P).

[0091] The present invention also concerns polynucleotide sequencesencoding polypeptide sequences of 5 or more amino acids, preferably 10or more amino acids, and up to the full length of the polypeptide,encoded by any one of ORFs 2-4 of VR 2431, in which the polynucleotidesat the codon(s) corresponding to the amino acid positions detailed inthe preceding three paragraphs are replaced with polynucleotidesencoding the corresponding amino acids of the proteins encoded by thecorresponding ORF of VR 2431.

[0092] In a further embodiment of the present invention, the polynucleicacid encodes one or more proteins, or antigenic regions thereof, of aPRRSV. Preferably, the present nucleic acid encodes at least oneantigenic region of a PRRSV membrane (envelope) protein. Morepreferably, the present polynucleic acid encodes a hypervariable regionfrom a ORF 5 PRRSV protein product (see the discussion below) or (b)contains at least one copy of the ORF-5 gene from a high virulence (hv)phenotype isolate of PRRSV (see the description of “hv phenotype” inU.S. application Ser. No. 08/301,435) and a sufficiently long fragment,region or sequence of at least one of ORF-2, ORF-3, ORF-4, ORF-5 and/orORF-6 from the genome of a PRRSV isolate to encode on antigenic regionof the corresponding protein(s) and effectively stimulate protectionagainst a subsequent challenge with, for example, a hv phenotype PRRSVisolate.

[0093] Even more preferably, at least one entire envelope proteinencoded by ORF-2, ORF-3, ORF-5 and/or ORF-6 of a PRRSV is contained inthe present polynucleic acid, and the present polynucleic acid excludesor modifies a sufficiently long portion of one of ORFs 2-4 from a PRRSVto render a PRRSV containing the same either low-virulent ornon-virulent. Most preferably, the polynucleic acid is isolated from thegenome of an isolate of the Iowa strain of PRRSV (for example, VR 2385(3× plaque-purified ISU-12), VR 2386 (non-plaque-purified ISU-12), VR2428 (ISU-51), VR 2429 (ISU-22), VR 2430 (ISU-55), VR 2431 (ISU-3927),VR 2474 (ISU-79) and/or ISU-1894).

[0094] A further preferred embodiment of the present invention includesa polynucleotide encoding an amino acid sequence from a hypervariableregion of ORF 5 of a PRRSV, preferably of an Iowa strain of PRRSV. Thus,such polynucleotides encode one (or more) of the following amino acidsequences: TABLE 1 Hypervariable Hypervariable Hypervariable Region 1Region 2 Region 3 (Positions 32-38) (Positions 57-66) (Positions120-128) NGNSGSN    (SEQ ID NO:45) ANKFDWAVET (SEQ ID NO:46) LICFVIRLA(SEQ ID NO:47) SNDSSSH    (SEQ ID NO:48) ANKFDWAVEP (SEQ ID NO:49)LTCFVIRFA (SEQ ID NO:50) SSSNSSH    (SEQ ID NO:51) AGEFDWAVET (SEQ IDNO:52) LICFVIRFT (SEQ ID NO:53) SANSSSH    (SEQ ID NO:54) ADKFDWAVEP(SEQ ID NO:55) LACFVIRFA (SEQ ID NO:56) HSNSSSH    (SEQ ID NO:57)ADRFDWAVEP (SEQ ID NO:58) LTCFVIRFV (SEQ ID NO:59) SNSSSSH    (SEQ IDNO:60) SSHFGWAVET (SEQ ID NO:61) LTCFIIRFA (SEQ ID NO:62)NNSSSSH    (SEQ ID NO:63) FICFVIRFA (SEQ ID NO:64) NGGDSST(Y) (SEQ IDNOS:65-66) FVCFVIRAA (SEQ ID NO:67)

[0095] In this embodiment, the polynucleotide may encode further aminoacid sequences of a PRRSV ORF 5 (as disclosed in FIG. 3 or in U.S.application Ser. Nos. 08/131,625 or 08/301,435), as long as one or moreof the hypervariable regions at positions 32-38, 57-66 and/or 120-128are included. (The present invention specifically excludes the proteinsand polynucleotides of ORF 5 of LV and VR 2332.)

[0096] A further preferred embodiment of the present invention concernsa purified preparation which may comprise, consist essentially of orconsist of a polynucleic acid having a sequence of the formula (I) or(II):

5′-α-β-3′  (I)

5′-α-β-γ-3′  (II)

[0097] wherein α encodes at least one polypeptide, or antigenic orlow-virulence fragment thereof encoded by a polynucleotide selected fromthe group consisting of ORFs 2, 3 and 4 of an Iowa strain of PRRSV andregions thereof encoding such antigenic and/or low-virulence fragments;and P is at least one copy of an ORF 5 from an Iowa strain of PRRSV oran antigenic fragment thereof (e.g. one or more hypervariable regions),preferably a full-length copy from a high replication (hr) phenotype;and γ encodes at least one polypeptide or antigenic fragment thereofencoded by a polynucleotide selected from the group consisting of ORF 6and ORF 7 of an Iowa strain of PRRSV and regions thereof encoding theantigenic fragments.

[0098] Alternatively, the present invention may concern a purifiedpreparation which may comprise, consist essentially of or consist of apolynucleic acid having a sequence of the formula (III):

5′-β-8-γ-3′  (III)

[0099] where β and γ are as defined above; and 8 is either a covalentbond or a linking polynucleic acid which does not materially affecttranscription and/or translation of the polynucleic acid. Preferably, βis a polynucleotide encoding at least one hypervariable region of aprotein encoded by an ORF 5 of an Iowa strain of PRRSV, and morepreferably, encodes a full-length protein encoded by an ORF 5 of an Iowastrain of PRRSV.

[0100] The present invention may also concern a purified preparationwhich may comprise, consist essentially of or consist of a polynucleicacid having a sequence of the formula (IV):

5′-α-β-δ-γ-3′  (IV)

[0101] where α, β, γ and δ are as defined in formulas (I)-(III) above.

[0102] The present invention may also concern a purified preparationwhich may comprise, consist essentially of or consist of a polynucleicacid, an expression vector or a plasmid having a sequence of the formula(V):

5′-ε-ζ-ι-τ-ξ-3′  (V)

[0103] where ε, which is optionally present, is a 5 ′-terminalpolynucleotide sequence which provides a means for operationallyexpressing the polynucleotides α, β, γ and δ; ζ is a polynucleotide ofthe formula KTVACC, where K is T, G or U, and V is A, G or C; ι is apolynucleotide of at most about 130 (preferably at most 100) nucleotidesin length; K is a polynucleotide comprising one or more genes selectedfrom the group consisting of a conventional marker or reporter gene, α,β, γ and operationally linked combinations thereof, where α, β, and γare as defined in formulas (I)-(IV) above; and ξ, which is optionallypresent, is a 3′-terminal polynucleotide sequence which does notsuppress the operational expression of the polynucleotides α, β, γ andδ, and which may be operationally linked to ε (for example, in aplasmid).

[0104] Suitable marker or reporter genes include, e.g., those providingresistance to an antibiotic such as neomycin, erythromycin orchloramphenicol; those encoding a known, detectable enzyme such asβ-lactamase, DHFR, horseradish peroxidase, glucose-6-phosphatedehydrogenase, alkaline phosphatase, and enzymes disclosed in U.S. Pat.No. 4,190,496, col. 32, line 33 through col. 38, line 44 (incorporatedherein by reference), etc.; and those encoding a known antibody (e.g.,mouse IgG, rabbit IgG, rat IgG, etc.) or known antigenic protein such asProtein A, Protein G, bovine serum albumin (BSA), keyhole limpethemocyanin (KLH), bovine gamma globulin (BGG), lactalbumin, polylysine,polyglutamate, lectin, etc.

[0105] The polynucleotide ι is preferably a polynucleotide sequence atleast 80% homologous to a polynucleotide sequence from a PRRSV genomelocated between a leader-mRNA junction sequence and the start codon ofthe ORF immediately downstream. “About 130” nucleotides in length refersto a length of the polynucleotide ι which does not adversely affect theoperational expression of τ. For example, in ISU 79, a leader-mRNAjunction sequence which does not suppress expression of ORF 7 can befound 129 bases upstream from the start codon of ORF 7 (see Experiment 2below). Suitable exemplary sequences for the polynucleotide can bededuced from the sequences shown in FIGS. 1 and 9.

[0106] The present polynucleic acid may also comprise, consistessentially of or consist of combinations of the above sequences, eitheras a mixture of polynucleotides or covalently linked in either ahead-to-tail (sense-antisense) or head-to-head fashion. Polynucleicacids complementary to the above sequences and combinations thereof(antisense polynucleic acid) are also encompassed by the presentinvention. Thus, in addition to possessing multiple or variant copies ofORF 5, the present polynucleic acid may also contain multiple or variantcopies of one or more of ORF's 1-7, including antigenic or hypervariableregions of ORF 5, of Iowa strain PRRSV′S.

[0107] Similar to the methods described above and in the Experimentsdescribed below and in U.S. application Ser. Nos. 08/131,625 and08/301,435, one can prepare a library of recombinant clones (e.g., usingE. coli as a host) containing suitably prepared restriction fragments ofa PRRSV genome (e.g., inserted into an appropriate plasmid expressiblein the host). The clones are then screened with a suitable probe (e.g,based on a conserved sequence of ORF's 2-3; see, for example, FIG. 22 ofU.S. application Ser. No. 08/301,435). Positive clones can then beselected and grown to an appropriate level. The polynucleic acids canthen be isolated from the positive clones in accordance with knownmethods. A suitable primer for PCR can then be designed and prepared asdescribed above to amplify the desired region of the polynucleic acid.The amplified polynucleic acid can then be isolated and sequenced byknown methods.

[0108] The present purified preparation may also contain a polynucleicacid selected from the group consisting of sequences having at least 97%sequence identity (or homology) with at least one of ORFs 5-7 of VR2385, VR 2430 and/or VR 2431; and sequences encoding a polypeptidehaving at least the minimum sequence identity (or homology) with atleast one of ORF's 2-5 of VR 2385, VR 2428, VR 2429, VR 2430, VR 2431,VR 2474 and ISU-1894, as follows: TABLE 2 Relative to Minimum % Homologywith ORF: Isolate: 2 3 4 5 VR 2385 99 92 95 90 VR 2429 100  99 99 98 VR2430 98 95 96 90 VR 2431 94 88 93 92 VR 2474 99 97 97 95 ISU 1894 97 9799 97

[0109] Preferably, the polynucleic acid excludes or modifies asufficiently long region or portion of one or more of ORFs 2-4 of the hvPRRSV isolates VR 2385, VR 2429, ISU-28, ISU-79 and/or ISU-984 to renderthe isolate low-virulent or non-virulent.

[0110] In the context of the present application, “homology” refers tothe percentage of identical nucleotide or amino acid residues in thesequences of two or more viruses, aligned in accordance with aconventional method for determining homology (e.g., the MACVECTOR orGENEWORKS computer programs, aligned in accordance with the proceduredescribed in Experiment III in U.S. application Ser. No. 08/301,435).

[0111] Preferably, the present isolated polynucleic acid encodes aprotein, polypeptide, or antigenic fragment thereof which is at least 10amino acids in length and in which non-homologous amino acids which arenon-essential for antigenicity may be conservatively substituted. Anamino acid residue in a protein, polypeptide, or antigenic fragmentthereof is conservatively substituted if it is replaced with a member ofits polarity group as defined below:

[0112] Basic Amino Acids:

[0113] lysine (Lys), arginine (Arg), histidine (His)

[0114] Acidic Amino Acids:

[0115] aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn),glutamine (Gln)

[0116] Hydrophilic, Nonionic Amino Acids:

[0117] serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn),glutamine (Gln)

[0118] Sulfur-Containing Amino Acids:

[0119] cysteine (Cys), methionine (Met)

[0120] Hydrophobic, Aromatic Amino Acids:

[0121] phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp)

[0122] Hydrophobic, Nonaromatic Acids:

[0123] glycine (Gly), alanine (Ala), valine (Val), leucine (Leu),isoleucine (Ile), proline (Pro)

[0124] More particularly, the present polynucleic acid encodes one ormore of the protein(s) encoded by the second, third, fourth, fifth,sixth and/or seventh open reading frames (ORF's 2-7) of the PRRSVisolates VR 2385, VR 2386, VR 2428, VR 2429, VR 2430, VR 2431, VR 2474and/or ISU-1894 (e.g., one or more of the sequences shown in FIG. 3and/or SEQ ID NOS:15, 17, 19, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63and 65 of U.S. application Ser. No. 08/301,435).

[0125] ORF's 6 and 7 are not likely candidates for controlling virulenceand replication phenotypes of PRRSV, as the nucleotide sequences ofthese genes are highly-conserved among high virulence (hv) and lowvirulence (lv) isolates (see Experiment III of U.S. application Ser. No.08/301,435). However, ORF 5 in PRRSV isolates appears to be lessconserved among high replication (hr) and low replication (1r) isolates.Therefore, it is believed that the presence of an ORF 5 from an hr PRRSVisolate in the present polynucleic acid will enhance the production andexpression of a recombinant vaccine produced from the polynucleic acid.

[0126] Furthermore, ORF 5 of PRRSV contains three hydrophilic,hypervariable regions typically associated with antigenicity in apolypeptide. Thus, the present invention also encompassespolynucleotides encoding a polypeptide comprising one or morehypervariable regions of a PRRSV ORF 5, preferably a polypeptide of theformula a-b-c-d-e-f-g, where:

[0127] a is an amino group, a poly(amino acid) corresponding topositions 1-31 of a protein encoded by a PRSSV ORF 5, or a fragment ofsuch a poly(amino acid) which does not adversely affect the antigenicityof the polypeptide;

[0128] b is an amino acid sequence selected from the group consisting ofthose sequences listed under Hypervariable Region No. 1 in Table 1above,

[0129] c is an amino acid sequence corresponding to positions 39-56 of aprotein encoded by a PRSSV ORF 5 (preferably a sequence of the formulaLQLIYNLTLCELNGTDWL (SEQ ID NO: 104), in which one or more [preferably1-10] amino acids may be conservatively substituted),

[0130] d is an amino acid sequence selected from the group consisting ofthose sequences listed under Hypervariable Region No. 2 in Table 1above,

[0131] e is an amino acid sequence corresponding to positions 67-119 ofa protein encoded by a PRRSV ORF 5, in which one or more (preferably1-20, and more preferably 1-10) amino acid residues may beconservatively substituted and which does not adversely affect theantigenicity of the polypeptide,

[0132] f is an amino acid sequence selected from the group consisting ofthose sequences listed under Hypervariable Region No. 3 in the Tableabove, and

[0133] g is a carboxy group (a group of the formula -COOH), an aminoacid sequence corresponding to positions 129-200 of a protein encoded bya PRSSV ORF 5 or a fragment thereof which does not adversely affect theantigenicity of the polypeptide.

[0134] Accordingly, it is preferred that the present polynucleic acid,when used for immunoprotective purposes (e.g., in the preparation of avaccine), contain at least one copy of ORF 5 from a high-replicationisolate (i.e., an isolate which grows to a titer of 10⁶-10⁷ TCID₅₀ in,for example, CRL 11171 cells; also see the discussions in ExperimentsVIII-XI U.S. application Ser. No. 08/301,435).

[0135] On the other hand, the lv isolate VR 2431 appears to be adeletion mutant, relative to hv isolates (see Experiments III andVIII-XI U.S. application Ser. No. 08/301,435). The deletion appears tobe in ORF 4, based on Northern blot analysis. Accordingly, when used forimmunoprotective purposes, the present polynucleic acid preferably doesnot contain a region of ORF 4 from an hv isolate responsible for highvirulence, and more preferably, excludes the region of ORF 4 which doesnot overlap with the adjacent ORF's 3 and 5.

[0136] It is also known (at least for PRRSV) that neither thenucleocapsid protein nor antibodies thereto confer immunologicalprotection against PRRSV to pigs. Accordingly, the present polynucleicacid, when used for immunoprotective purposes, contains one or morecopies of one or more regions from ORF's 2, 3, 4, 5 and 6 of a PRRSVisolate encoding an antigenic region of the viral envelope protein, butwhich does not result in the symptoms or histopathological changesassociated with PRRS when administered to a pig. Preferably, this regionis immunologically cross-reactive with antibodies to envelope proteinsof other PRRSV isolates.

[0137] Similarly, the protein encoded by the present polynucleic acidconfers protection against PRRS to a pig administered a compositioncomprising the protein, and antibodies to this protein areimmunologically cross-reactive with the envelope proteins of other PRRSVisolates. More preferably, the present polynucleic acid encodes theentire envelope protein of a PRRSV isolate or a protein at least 80%homologous thereto and in which non-homologous residues areconservatively substituted, or alternatively a protein at least 98%homologous thereto. Most preferably, the present polynucleotide is oneof the sequences shown in FIG. 1, encompassing at least one of the openreading frames recited therein.

[0138] Relatively short segments of polynucleic acid (about 20 bp orlonger) in the genome of a virus can be used to screen or identifytissue and/or biological fluid samples from infected animals, and/or toidentify related viruses, by methods described herein and known to thoseof ordinary skill in the fields of veterinary and viral diagnostics andveterinary medicine. Accordingly, a further aspect of the presentinvention encompasses an isolated (and if desired, purified) polynucleicacid consisting essentially of a fragment of from 15 to 2000 bp,preferably from 18 to 1000 bp, and more preferably from 21 to 100 bp inlength, derived from ORF's 2-7 of a PRRSV genome (preferably the Iowastrain of PRRSV). Particularly preferably, the present isolatedpolynucleic acid fragments are obtained from a terminus of one or moreof ORF′ s 2-7 of the genome of the Iowa strain of PRRSV, and mostpreferably, are selected from the group consisting of the primersdescribed in Experiments 1 and 2 below and SEQ ID NOS: 1-12, 22 and28-34 of U.S. application Ser. No. 08/301,435.

[0139] The present invention also concerns a diagnostic kit for assayinga porcine reproductive and respiratory syndrome virus, comprising (a) afirst primer comprising a polynucleotide having a sequence of from 10 to50 nucleotides in length which hybridizes to a genomic polynucleic acidfrom an Iowa strain of porcine reproductive and respiratory syndromevirus at a temperature of from 25 to 75° C., (b) a second primercomprising a polynucleotide having a sequence of from 10 to 50nucleotides in length, said sequence of said second primer being foundin said genomic polynucleic acid from said Iowa strain of porcinereproductive and respiratory syndrome virus and being downstream fromthe sequence to which the first primer hybridizes, and (c) a reagentwhich enables detection of an amplified polynucleic acid. Preferably,the reagent is an intercalating dye, the fluorescent properties of whichchange upon intercalation into double-stranded DNA.

[0140] The present isolated polynucleic acid fragments can be obtainedby: (i) digestion of the cDNA corresponding to (complementary to) theviral polynucleic acids with one or more appropriate restrictionenzymes, (ii) amplification by PCR (using appropriate primerscomplimentary to the 5′ and 3′-terminal regions of the desired ORF(S) orto regions upstream of the 5′-terminus or downstream from the3′-terminus) and cloning, or (iii) synthesis using a commerciallyavailable automated polynucleotide synthesizer.

[0141] Another embodiment of the present invention concerns one or moreproteins or antigenic fragments thereof from a PRRS virus, preferablyfrom the Iowa strain of PRRSV. As described above, an antigenic fragmentof a protein from a PRRS virus (preferably from the Iowa strain ofPRRSV) is at least 5 amino acids in length, particularly preferably atleast 10 amino acids in length, and provides or stimulates animmunologically protective response in a pig administered a compositioncontaining the antigenic fragment.

[0142] Methods of determining the antigenic portion of a protein areknown to those of ordinary skill in the art (see the description above).In addition, one may also determine an essential antigenic fragment of aprotein by first showing that the full-length protein is antigenic in ahost animal (e.g., a pig). If the protein is still antigenic in thepresence of an antibody which specifically binds to a particular regionor sequence of the protein, then that region or sequence may benon-essential for immunoprotection. On the other hand, if the protein isno longer antigenic in the presence of an antibody which specificallybinds to a particular region or sequence-of the protein, then thatregion or sequence is considered to be essential for antigenicity.

[0143] Three hypervariable regions in ORF 5 of PRRSV have beenidentified by comparing the amino acid sequences of the ORF 5 product ofall available PRRSV isolates (see, for example, FIG. 2D). Amino acidvariations in these three regions are significant, and are notstructurally conserved (FIG. 2D). All three hypervariable regions arehydrophilic and antigenic. Thus, these regions are likely to be exposedto the viral membrane and thus be under host immune selection pressure.

[0144] The present invention also concerns a protein or antigenicfragment thereof encoded by one or more of the polynucleic acids definedabove, and preferably by one or more of the ORF's of a PRRSV, morepreferably of the Iowa strain of PRRSV. The present proteins andantigenic fragments are useful in immunizing pigs against PRRSV, inserological tests for screening pigs for exposure to or infection byPRRSV (particularly the Iowa strain of PRRSV), etc.

[0145] For example, the present protein may be selected from the groupconsisting of the proteins encoded by ORF's 2-7 of VR 2385, ISU-22 (VR2429), ISU-55 (VR 2430), ISU-1894, ISU-79 (VR 2474) and ISU-3927 (VR2431) (e.g., one or more of the sequences shown in FIG. 2 and/or SEQ IDNOS: 15, 17, 19, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 67, 69 and 71of U.S. application Ser. No. 08/301,435); antigenic regions of at leastone of these proteins having a length of from 5 amino acids to less thanthe full length of the protein; polypeptides having the minimum homologywith the protein encoded by the PRSSV ORF indicated in Table 2 above;and polypeptides at least 97% homologous with a protein encoded by oneof the ORF's 6-7 of VR 2385, VR 2429, VR 2430, ISU-1894, ISU-79 and VR2431 (e.g., SEQ ID NOS:17, 19, 43, 45, 47, 49, 51, 53, 55, 57, 59 and 61of U.S. application Ser. No. 08/301,435). Preferably, the presentprotein has a sequence encoded by an ORF selected from the groupconsisting of ORFs 2-5 of VR 2385, VR 2428, VR 2429, VR 2430, VR 2431,VR 2474 and ISU-1894 (see, for example, FIGS. 2A-D); variants thereofwhich provide effective immunological protection to a pig administeredthe same and in which from 1 to 100 (preferably from 1 to 50 and morepreferably from 1 to 25) deletions or conservative substitutions in theamino acid sequence exist; and antigenic fragments thereof at least 5and preferably at least 10 amino acids in length which provide effectiveimmunological protection to a pig administered the same.

[0146] More preferably, the present protein variant or protein fragmenthas a binding affinity (or association constant) of at least 1% andpreferably at least 10% of the binding affinity of the correspondingfull-length, naturally-occurring protein to a monoclonal antibody whichspecifically binds to the full-length, naturally-occurring protein(i.e., the protein encoded by a PRRSV ORF).

[0147] The present invention also concerns a method of producing apolypeptide, comprising expressing the present polynucleic acid in anoperational expression system, and purifying the expressed polypeptidefrom the expression system. Suitable expression systems include thoseconventionally used for either in vitro or in vivo expression ofproteins and polypeptides, such as a rabbit reticulocyte system for invitro expression, and for in vivo expression, a modified or chimericPRRSV (used to infect an infectable host cell line, such as MA-104, CRL11171, PSP-36, PSP-36-SAH, MARC-145 and porcine alveolar macrophages),or a conventional expression vector containing the present polynucleicacid, under the operational control of a known promoter (e.g., athymidine kinase promoter, SV40, etc.) for use in conventionalexpression systems (e.g., bacterial plasmids and corresponding hostbacteria, yeast expression systems and corresponding host yeasts, etc.).The expressed polypeptide or protein is then purified or isolated fromthe expression system by conventional purification and/or isolationmethods.

[0148] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments, which aregiven for illustration of the invention, and are not intended to belimiting thereof.

EXAMPLE 1

[0149] Summary:

[0150] The sequences of ORFs 2 to 5 of one low virulence, one “moderate”virulence and one high virulence U.S. PRRSV isolate have been determinedand analyzed. Comparisons with known sequences of other PRRSV isolatesshow that considerable sequence variations at both nucleotide and aminoacid levels exist in ORFs 2 to 5 of seven U.S. isolates with differingvirulence. However, ORFs 6 and 7 of these seven U.S. isolates are highlyconserved (U.S. application Ser. No. 08/301,435). Extensive sequencevariations were also found in ORFs 2 to 7 between the European LV andthe U.S. isolates. The least virulent U.S. PRRSV isolate known(ISU-3927) displayed the most sequence variation, in comparison withother U.S. isolates.

[0151] The phylogenetic relationship of the U.S. isolates was alsoanalyzed. Phylogenetic analysis of the ORFs 2 to 7 of the U.S. isolatesindicated that there are at least three groups of PRRSV variants (orminor genotypes) within the major U.S. PRRSV genotype. Consequently, itis highly likely that a number of additional major or minor genotypeswill be identified as more virus isolates from different geographicregions are examined.

[0152] Interestingly, the least virulent U.S. isolate known (ISU 3927)forms a branch distinct from other U.S. isolates. Analysis of thenucleotide and amino acid sequences also showed that the isolate ISU3927 exhibits the most variations in ORFs 2 to 4, relative to other U.S.isolates. Many of these variations in isolate ISU 3927 result innon-conserved amino acid substitutions. However, these non-conservedchanges in isolate ISU 3927, as compared to other U.S. isolates, do notappear to be limited to a particular region; they are present throughoutORFs 2 to 4. Therefore, a specific correlation between sequencevariations and viral virulence is not yet fully elucidated (althoughcertain positions in ORF 3 appear to be possibly related to virulence;see FIG. 2B, positions 30, 48, 54-56, 134, 140, 143, 147, 153, 206, and215; amino acids at one or more of these positions may serve as a basisfor mutating other known proteins encoded by a PRRSV ORF 3).

[0153] Results:

[0154] The amino acid sequence identity between seven U.S. PRRSVisolates was 91-99% in ORF 2, 86-98% in ORF 3, 92-99% in ORF 4 and88-97% in ORF 5. The least virulent U.S. isolate known has highersequence variations in the ORFs 2 to 4 than in ORFs 5 to 7, as comparedto other U.S. isolates. Three hypervariable regions with antigenicpotential were identified in the major envelope glycoprotein encoded byORF 5.

[0155] Pairwise comparison of the sequences of ORFs 2 to 7 andphylogenetic tree analysis implied the existence of at least threegroups of PRRSV variants (or minor genotypes) within the major genotypeof U.S. PRRSV. The least virulent U.S. isolate known forms a distinctbranch from other U.S. isolates with differing virulence. The results ofthis study have implications for the taxonomy of PRRSV and vaccinedevelopment.

[0156]FIG. 1 shows a nucleotide sequence comparison of ORFs 2 to 5 ofU.S. isolates ISU 3927, ISU 22 and ISU 55 with other known PRRSVisolates. The nucleotide sequence of VR 2385 is shown on top, and onlydifferences are indicated. The start codon of each ORF is indicated by+>, and the termination codon of each ORF is indicated by asterisks (*).The leader-niRNA junction sequences for subgenomic mRNAs 3, 4 and 4-1are underlined, and the locations of the junction sequences relative tothe start codon of each ORF are indicated by minus (−) numbers ofnucleotides upstream of each ORF. The sequences of VR 2385 (U.S.application Ser. Nos. 08/131,625 and 08/301,435), VR 2332, ISU 79 andISU 1894 (U.S. application Ser. No. 08/301,435) used in this alignmentwere previously reported.

[0157] Materials and Methods:

[0158] Cells and Viruses:

[0159] The ATCC CRL 11171 cell line was used to propagate the PRRSV. Thecells were grown in Dulbecco′ s minimal essential medium (DMEM)supplemented with 10% fetal bovine serum (FBS) and 1× antibiotics(penicillin G 10,000 unit/ml, streptomycin 10,000 mg/ml and amphotericinB 25 mg/ml).

[0160] Three U.S. isolates of PRRSV used in this study, designated asISU 22, ISU 55 and ISU 3927, were isolated from pig lungs obtained fromdifferent farms in Iowa during PRRS outbreaks. All three isolates wereplaque-purified three times on CRL 11171 cells before furtherexperimentation. Comparative pathogenicity studies showed that isolateISU 3927 is the least virulent isolate among 10 different U.S. PRRSVisolates. Isolate ISU 22 is a high virulence isolate and isolate ISU 55is “moderately” pathogenic. All of the three virus isolates used in thisexperiment were at seventh passage.

[0161] Isolation of PRRSV Intracellular RNAs:

[0162] Confluent monolayers of CRL 11171 cells were infected with thethree U.S. isolates of PRRSV, ISU 22, ISU 55 and ISU 3927, respectively,at a multiplicity of infection (m.o.i.) of 0.1. At 24 hrs.postinfection, the infected cells were washed three times with cold PBSbuffer. The total intracellular RNAs were then isolated by guanidiniumisothiocyanate and phenol-chloroform extraction (Stratagene). Thepresence of virus-specific RNA species in the RNA preparation wasconfirmed by Northern blot hybridization (data not shown). The totalintracellular RNAs were quantified spectrophotometrically.

[0163] Reverse transcription and polymerase chain reaction (RTPCR):

[0164] First strand complementary (c) DNA was synthesized from the totalintracellular RNAs by reverse transcription using random primers asdescribed previously (Meng et al., 1993, J. Vet. Diagn. Invest.,5:254-258). For amplification of the entire protein coding regions ofthe ORFs 2 to 5 of the three isolates of PRRSV, two sets of primers weredesigned on the basis of the sequences of VR 2385 and LV. Primers JM259(5′-GGGGATCCTTTTGTGGAGCCGT-3′; SEQ ID NO:68) and JM260(5′-GGGGAATTCGGGATAGGGAATGTG-3′; SEQ ID NO:69) amplified the sequence ofORFs 4 and 5, and primers XM992 (5′-GGGGGATCCTGTTGGTAATAG(A)GTCTG-3′(SEQ ID NOS:70-71) and XM993 (5′-GGTGAATTCGTTTTATTTCCCTCCGGGC-3′; SEQ IDNO:72) amplified the sequence of ORFs 2 and 3. Unique restriction sites(EcoRI or BamHI) at the 5′ end of these primers were introduced tofacilitate cloning. A degenerate base, G (A), was synthesized in primerXM 992 based on the sequences of VR 2385 and LV (Meulenberg et al.,1993; U.S. application Ser. No. 08/301,435). PCR was performed asdescribed previously (Meng et al., 1993, J. Vet. Diagn. Invest.,5:254-258).

[0165] Cloning and Nucleotide Sequencing:

[0166] The RT-PCR products were analyzed by a 0.8% agarose gelelectrophoresis. The two PCR fragments representing ORFs 2 and 3 as wellas ORFs 4 and 5, respectively, were purified by the glassmilk procedure(GENECLEAN kit, BIO 101, Inc.). The purified fragments were eachdigested with BamHI and EcoRI, and cloned into the vector pSK+ asdescribed previously (Meng et al., 1993). The E. coli DH 5α cells wereused for transformation of recombinant plasmids. White colonies wereselected and grown in LB broth containing 100 mg/ml ampicillin. The E.coli cells containing recombinant plasmid were lysed with lysozyme, andthe plasmids were then isolated by using the Qiagen column (QIAGENInc.).

[0167] Plasmids containing viral inserts were sequenced with anautomated DNA Sequencer (Applied Biosystem, Inc.). Three or moreindependent cDNA clones representing the entire sequence of ORFs 2 to 5from each of the three PRRSV isolates were sequenced with universal andreverse primers. Several virus-specific primers, XM969(5′-GATAGAGTCTGCCCTTAG-3′; SEQ ID NO:73), XM970(5′-GTTTCACCTAGAATGGC-3′; SEQ ID NO:74), XM1006(5′-GCTTCTGAGATGAGTGA-3′; SEQ ID NO:75), XM077(5′-CAACCAGGCGTAAACACT-3′; SEQ ID NO:76) and XM078 (5′-CTGAGCAATTACAGAAG-3′; SEQ ID NO:77), were also used to determine the sequence ofORFs 2 to 5.

[0168] Sequence Analyses:

[0169] Sequence data were combined and analyzed by using MacVector(International Biotechnologies, Inc.) and GeneWorks (IntelliGenetics,Inc.) computer software programs. Phylogenetic analyses were performedusing the PAUP software package version 3.1.1 (David L. Swofford,Illinois Natural History Survey, Champaign, Ill.). PAUP employs themaximum parsimony algorithm to construct phylogenetic trees.

[0170] Results:

[0171] Nucleotide Sequence Analyses of ORFs 2 to 5:

[0172] The sequences of ORFS 2 to 5 of five PRRSV isolates, ISU 79, ISU1894, ISU 22, ISU 55 and ISU 3927, were determined and compared withother known PRRSV isolates including VR 2385, VR 2332 and LV (Meulenberget al., 1993). The sequences of ORFs 6 and 7 of isolates VR 2385, ISU22, ISU 55, ISU 79, ISU 1894 and ISU 3927 were reported previously (U.S.application Ser. No. 08/301,435). The isolates used in this experimenthave been shown to differ in pneumovirulence in experimentally-infectedpigs (U.S. application Ser. Nos. 08/131,625 and 08/301,435). ISU 3927 isthe least virulent isolate among ten different U.S. PRRSV isolates (U.S.application Ser. No. 08/131,625 and U.S. application Ser. No.08/301,435).

[0173] Like other U.S. PRRSV isolates, ORFs 2 to 4 of these isolatesoverlapped each other (FIG. 1). However, unlike LV, ORFs 4 and 5 of theU.S. isolates are separated by 10 nucleotides (FIG. 1). ORFs 4 and 5 ofIV overlapped by one nucleotide. The single nucleotide substitution fromA of the start codon of ORF 5 in LV to T in the U.S. isolates places thestart codon of ORF 5 of the U.S. isolates 10 nucleotides downstream ofthe ORF 4 stop codon. Therefore, a 10-nucleotide noncoding sequenceappears between ORFs 4 and 5 of the known U.S. isolates (FIG. 1).

[0174] ORF 2 of ISU 79 is 3 nucleotides shorter than other U.S.isolates. The single nucleotide substitution from TGG to TAG just beforethe stop codon of ORF 2 creates a new stop codon in ISU 79 (FIG. 1). A3-nucleotide deletion was also found in ORF 5 of ISU 3927, compared toother U.S. isolates (FIG. 1). The size of ORFs 2 to 5 of all the U.S.isolates are identical, except for the ORF 2 of ISU 79 and ORF 5 of ISU3927, both of which are 3 nucleotides shorter than the other ORFs (FIG.1).

[0175] Sequence comparisons of ORFs 2 to 5 of the seven U.S. PRRSVisolates shown in FIG. 1 indicate that there are considerable nucleotidesequence variations in ORFs 2 to 5 of the U.S. isolates (FIG. 1). Thenucleotide sequence identity was 96-98% in ORF 2, 92-98% in ORF 3,92-99% in ORF 4, and 90-98% in ORF 5 between VR 2385, VR 2332, ISU 22,ISU 55, ISU 79, and ISU 1894 (Table 3).

[0176] The least virulent isolate ISU 3927 has the most variations amongthe seven U.S. isolates (FIG. 1 and Table 3). The nucleotide sequenceidentity between ISU 3927 and other U.S. isolates was 93-94% in ORF 2,89-90% in ORF 3, and 91-93% in ORF 4 (Table 3). Like ORFs 6 and 7 (U.S.application Ser. No. 08/301,435), ORF 5 of ISU 3927 has no significantchanges except for a 3-nucleotide deletion (FIG. 1). ORF 5 of ISU 3927shares 91-93% nucleotide sequence identity with the ORF 5 of other U.S.isolates (Table 3).

[0177] However, extensive sequence variation was found in ORFS 2 to 5between LV and the U.S. isolates (FIG. 1 and Table 3). The nucleotidesequence identity between LV and the U.S. isolates was 65-67% in ORF 2,61-64% in ORF 3, 63-66% in ORF 4, and 61-63% in ORF 5 (Table 3).Extensive genetic variations in ORFs 6 and 7 between LV and U.S. PRRSValso exist (U.S. application Ser. Nos. 08/131,625 and 08/301,435). Theseresults indicate that the least virulent isolate ISU 3927 is also themost distantly related of the U.S. isolates, with genetic variationsoccurring mostly in ORFs 2 to 4.

[0178] The single nucleotide substitution from TGG to TAG before thestop codon in ORF 2 observed in ISU 79 was also present in isolates ISU55 and ISU 3927, both of which produce seven sg niRNAs, but not inisolates ISU 22, ISU 1894 or VR 2385, which each synthesize only six sgmRNAs (U.S. application Ser. Nos. 08/131,625 and 08/301,435). Theresults indicate that the leader-mRNA 4-1 junction sequence of ISU 55and ISU 3927 is very likely to be the same as ISU 79 (FIG. 1).

[0179] The leader-mRNA junction sequences for sg mRNAs 3 and 4 of ISU 79and ISU 1894 were determined to be GUAACC at 89 nucleotides upstream ofORF 3 for sg mRNA 3, and UUCACC at 10 nucleotides upstream of ORF 4 forsg mRNA 4 (U.S. application Ser. No. 08/301,435; see also Experiment 2below). A sequence comparison of isolates ISU 22, ISU 55 and ISU 3927with isolates VR 2385, ISU 79 and ISU 1894 indicates that theleader-mRNA junction sequences for sg mRNAs 3 and 4 are conserved amongthe U.S. isolates (FIG. 1). Analysis of the deduced amino acid sequencesencoded by ORFs 2 to 5:

[0180]FIG. 2 shows the alignment of the deduced amino acid sequences ofORF 2 (A), ORF 3 (B), ORF 4 (C) and ORF 5 (D) of U.S. isolates ISU 22,ISU 55 and ISU 3927 with other known PRRSV isolates. The sequence of VR2385 is shown on top, and only differences are indicated. Deletions areindicated by (−). The proposed signal peptide sequence in the ORF 5 ofLV (D) is underlined (Meulenberg et al., 1995). Three hypervariableregions with antigenic potentials in ORF 5 (D) were indicated byasterisks (*). The published sequences used in this alignment were LV(Meulenberg et al., 1993), VR 2385 (U.S. application Ser. Nos.08/131,625 and 08/301,435), VR 2332, ISU 79 and ISU 1894 (U.S.application Ser. No. 08/301,435).

[0181] On the basis of its high content of basic amino acids and itshydrophilic nature, the translation product of ORF 7 is predicted to bethe nucleocapsid protein (U.S. application Ser. Nos. 08/131,625 and08/301,435; Meulenberg et al., 1993; Conzelmann et al., 1993; Mardassiet al., 1994). The ORF 6 product lacks a potential amino-terminal signalsequence and contains several hydrophobic regions which may representthe potential transmembrane fragments. Therefore, the ORF 6 product waspredicted to be the M protein (U.S. application Ser. Nos. 08/131,625 and08/301,435; Meulenberg et al., 1993; Conzelmann et al., 1993).

[0182] Computer analysis shows that the products encoded by ORFs 2 to 5of the U.S. isolates all have hydropathy characteristics reminiscent ofmembrane-associated proteins. The translation products of ORFs 2 to 5each contain a hydrophobic amino terminus. The N-terminal hydrophobicsequences may function as a signal sequence for each of these ORFS, andthey may be involved in the transportation of ORFs 2 to 5 to theendoplasmic reticulum of infected cells. At least one additionalhydrophobic domain in each of ORFs 2 to 5 was found at the carboxytermini. These additional hydrophobic domains may function as membraneanchors.

[0183] The deduced amino acid sequences of ORFs 2 to 5 of the seven U.S.isolates examined also varied considerably (FIG. 2), indicating thatmost of the nucleotide differences observed in FIG. 1 are not silentmutations. The amino acid sequence identity between VR 2385, VR 2332,ISU 22, ISU 55, ISU 79, and ISU 1894 was 95-99% in ORF 2, 90-98% in ORF3, 94-98% in ORF 4, and 88-97% in ORF 5 (Table 3).

[0184] Again, the least virulent isolate ISU 3927 displayed morevariations with other U.S. isolates in ORFs 2 to 4 (FIG. 2 and Table 3)than in ORFs 5 to 7 (U.S. application Ser. No. 08/301,435 and Table 3).ORFs 2 to 5 of LV share only 57-61%, 55-56%, 65-67%, and 51-55% aminoacid sequence identity with those ORFs of the U.S. isolates,respectively (Table 3). Deletions or insertions were found throughoutORFs 2 to 5 in comparing European IV and U.S. isolates (FIG. 2).

[0185] Sequence comparison of the ORF 5 product showed that theN-terminal region of ORF 5 is extremely variable, both (a) between U.S.isolates and LV and also (b) among the various U.S. isolates (FIG. 2D).In LV, the first 32-33 amino acid residues of ORF 5 may represent thesignal sequence (Meulenberg et al., 1995; FIG. 2D). Therefore, thepotential signal sequence of ORF 5 in all the PRRSV isolates is veryheterogeneous. This heterogeneity is not due to any host immuneselection pressure, because the signal peptide will be cleaved out andnot be present in mature virions.

[0186] Three additional hypervariable regions were also identified bycomparing the amino acid sequences of ORF 5 of all the PRRSV isolatesavailable (FIG. 2D). Amino acid variations in these three regions aresignificant, and are not structurally conserved (FIG. 2D). Computeranalysis indicates that all three hypervariable regions are hydrophilicand antigenic. Thus, it is likely that these regions are exposed to theviral membrane and are under host immune selection pressure. However,further experiments may be necessary to confirm the specific functionsof these hypervariable regions as antigenic determinants in the ORF 5envelope protein.

[0187] The Phylogenetic Relationships Among U.S. Isolates of PRRSV:

[0188] It has been shown previously that U.S. PRRSV and European PRRSVrepresent two distinct genotypes, based on analysis of the M and N genes(U.S. application Ser. No. 08/301,435). To determine the phylogeneticrelationships of U.S. PRRSV isolates, ORFs 2 to 7 of the seven U.S.PRRSV isolates shown in FIGS. 1 and 2 were first aligned with theGeneWorks program (intelligenetics, Inc.). The PAUP program (David L.Swofford, Illinois Natural History Survey, Champaign, Ill.) was thenused to construct phylogenetic tree illustrating relationship among U.S.isolates of PRRSV.

[0189] The phylogenetic tree of FIG. 3 was constructed by maximumparsimony methods with the aid of the PAUP software package version3.1.1. The branch with the shortest length (most parsimonious) was foundby implementing the exhaustive search option. The branch lengths(numbers of amino acid substitutions) are given above each branch. Thesequences used in the analysis are LV, VR 2385, VR 2332, ISU 79 and ISU1894.

[0190] The phylogenetic tree indicates that at least three groups ofvariants (or minor genotypes) exist within the major U.S. PRRSVgenotype. The least virulent U.S. PRRSV isolate ISU 3927 forms a branchdistinct from other U.S. isolates (FIG. 3). Isolates ISU 22, ISU 79, ISU1894, and VR 2332 form another branch, representing a second minorgenotype. The third minor genotype is represented by isolates ISU 79 andVR 2385 (FIG. 3). A very similar tree was also obtained by analyzing thelast 60 nucleotides of ORF 1b of the seven U.S. isolates presented inFIG. 1 (data not shown). Identical tree topology was also produced bythe unweighted pair-group method with arithmetic mean (UPGMA) using theGeneWorks program (data not shown).

[0191] In summary, the different genotypes of PRRSV have been confirmedand further elucidated. At least three minor genotypes within the majorgenotype of U.S. PRRSV have been identified, based on an analysis of thesequence of ORFs 2 to 7. Genetic variations not only between theEuropean PRRSV and the U.S. PRRSV but among the U.S. PRRSV isolates havealso been further confirmed as well, indicating the heterogeneous natureof PRRSV. The least virulent U.S. PRRSV isolate ISU 3927 hasunexpectedly high sequence variations in ORFs 2 to 4, as compared toother U.S. isolates. TABLE 3 Nucleotide and deduced amino acid sequenceidentities (%) of ORFs 2 to 5 of PRRSV VR2385 ISU22 ISU55 ISU79 ISU1894ISU3927 VR2332 LV ORF 2 VR2385 ** 97 96 96 95 91 98 58 ISU22 97 ** 96 9896 93 99 59 ISU55 98 97 ** 96 95 91 97 61 ISU79 96 97 97 ** 96 91 98 60ISU1894 96 97 96 96 ** 93 96 57 ISU3927 94 94 94 93 93 ** 93 58 VR233297 98 97 98 97 94 ** 59 LV 65 66 66 67 66 65 66 ** ORF 3 VR2385 ** 91 9492 90 87 91 55 ISU22 92 ** 93 96 96 88 98 56 ISU55 94 93 ** 94 93 87 9456 ISU79 94 96 94 ** 95 87 96 56 ISU1894 92 97 93 96 ** 86 96 55 ISU392790 90 89 90 90 ** 87 55 VR2332 93 98 94 97 97 90 ** 56 LV 64 63 62 63 6361 63 ** ORF 4 VR2385 ** 94 96 94 95 83 94 66 ISU22 93 ** 94 97 99 93 9866 ISU55 96 94 ** 96 96 93 95 67 ISU79 93 97 94 ** 98 92 96 66 ISU189492 98 94 96 ** 93 98 66 ISU3927 91 93 92 91 91 ** 92 67 VR2332 94 99 9597 98 92 ** 65 LV 66 66 63 65 66 65 65 ** ORF 5 VR2385 ** 90 91 88 89 9189 54 ISU22 93 ** 90 94 96 92 97 52 ISU55 94 92 ** 89 89 90 89 51 ISU7991 95 91 ** 95 89 94 53 ISU1894 92 97 90 94 ** 91 96 53 ISU3927 91 93 9191 91 ** 91 55 VR2332 93 98 91 95 97 92 ** 53 LV 63 63 63 61 62 63 63 **

EXAMPLE 2

[0192] During the replication of PRRSV, six subgenomic mRNAs (sg mRNAs),in addition to the genomic RNA, are synthesized. These sg mRNAs werecharacterized in this experiment.

[0193] The sg mRNAs of PRRSV form a 3 ′-coterminal nested set inPRRSV-infected cells. Each of these sg mRNAs is polycistronic andcontains multiple open reading frames, except for sg mRNA 7 (as shown byNorthern blot analysis using ORF-specific probes). The sg mRNAs were notpackaged into virions, and only the genomic RNA was detected in purifiedvirions, suggesting that the encapsulation signal of PRRSV is likelylocalized in the ORF 1 region.

[0194] The numbers of sg mRNAs in PRRSV-infected cells varies amongPRRSV isolates with differing virulence. An additional species of sgmRNA in some PRRSV isolates was shown in Experiment 1 above to bederived from the sequence upstream of ORF 4, and has been designated assg mRNA 4-1.

[0195] The leader-mRNA junction sequences of sg mRNAs 3 and 4 ofisolates ISU 79 and ISU 1894, as well as sg MRNA 4-1 of the isolate ISU79, contain a common six nucleotide sequence motif, T(G)TA(G/C)ACC.Sequence analysis of the genomic RNA of these two U.S. isolates andcomparison with Lelystad virus (IV) revealed heterogeneity of theleader-mRNA junction sequences among PRRSV isolates. The numbers,locations and the sequences of the leader-mRNA junction regions variedbetween U.S. isolates and LV, as well as among U.S. isolates. The lastthree nucleotides, ACC, of the leader-mRNA junction sequences areinvariable.

[0196] By comparing the 5′-terminal sequence of sg mRNA 4-1 with thegenomic sequence of ISU 79 and ISU 1894, it was found that a singlenucleotide substitution, from T in ISU 1894 to C in ISU 79, led to a newleader-mRNA junction sequence in ISU 79, and therefore, an additionalspecies of sg mRNA (sg mRNA 4-1). A small ORF, designated as ORF 4-1,with a coding capacity of 45 amino acids was identified at the 5′-end ofsg mRNA 4-1.

[0197] Materials and Methods

[0198] Viruses and Cells.

[0199] The PRRSV isolates used (ISU 22, ISU 55, ISU 79, ISU 1894 and ISU3927) were isolated from pig lungs obtained from different farms inIowa. A continuous cell line, ATCC CRL 11171, was used for isolation andgrowth (culturing) of viruses. These PRRSV isolates were biologicallycloned by three rounds of plaque purification and grown on the CRL 11171cells. All of the virus isolates used in this study were at the seventhpassage.

[0200] ISU 22 and ISU 79 are highly pathogenic and produce from 50 to80% consolidation of the lung tissues in experimentally-infectedfive-week-old caesarean-derived colostrum-deprived pigs necropsied at 10days post-inoculation. By contrast, ISU 55, ISU 1894 and ISU 3927 are oflow pathogenicity and produce only 10 to 25% consolidation of lungtissues in the same experiment (U.S. application Ser. Nos. 08/131,625and 08/301,435).

[0201] Preparation of virus-specific total intracellular RNAS, poly (A)⁺RNA and virion RNA. Confluent monolayers of CRL 11171 cells wereinfected with different isolates of PRRSV at the seventh passage at amultiplicity of infection (m.o.i.) of 0.1. PRRSV-specific totalintracellular RNAs were isolated from PRRSV-infected cells by aconventional guanidinium isothiocyanate method (Stratagene). The poly(A)⁺ RNA was enriched from the total intracellular RNAs by oligo(dT)-cellulose column chromatography (Invitrogen).

[0202] For isolation of PRRSV virion RNA, confluent CRL 11171 cells wereinfected with isolate ISU 3927 of PRRSV at a m.o.i. of 0.1. When morethan 70% of the infected cells showed a cytopathic effect, the cultureswere frozen and thawed three times, and the culture medium was clarifiedat 1200× g for 20 min. at 4° C. The virus was then precipitated withpolyethylene glycol and subsequently purified by cesium chloridegradient centrifugation as described in U.S. application Ser. No.08/131,625. The purified virus was treated with RNase A at a finalconcentration of 20 μ/ml for 90 min. at 37° C. The virus was thenpelleted, and the virion RNA was isolated using a conventionalguanidinium isothiocyanate method.

[0203] cDNA synthesis and polymerase chain reaction. cDNA wassynthesized from total intracellular RNAs by reverse transcription usingrandom primers and amplified by the polymerase chain reaction (RT-PCR)as described previously (Meng et al., 1993, J. Vet. Diagn. Invest.,5:254-258).

[0204] Northern blot analyses. Ten μg of total intracellular RNAs fromvirus infected cells and mock-infected cells were used per lane in aformaldehyde-agarose gel. For separation of poly (A)⁺ RNA and virionRNA, fifteen ng of virion RNA and 0.2 μg of poly (A)⁺ RNA were loadedper lane. The RNA was denatured with formaldehyde according to aconventional method (Sambrook et al, “Molecular Cloning: A LaboratoryManual”, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NewYork). Electrophoretic separation of RNA, RNA blotting, andhybridization were performed as described in U.S. application Ser. No.08/131,625. In some experiments, glyoxal-DMSO agarose gels were alsoperformed as described in U.S. application Ser. No. 08/131,625.

[0205] For preparation of probes, a specific cDNA fragment from each ofthe ORFs 1b to 7 was generated by RT-PCR with ORF-specific primers. Theprimers were designed in such a way that each primer pair amplifies onlya specific fragment of a given ORF, and the overlapping, neighboringORFs are not included in any given cDNA probe. The primer pairs forgenerating cDNA probes representing ORFs 1b through 7 are IM729/IM782for ORF lb, IM312/IM313 for ORF 2, XM1022/IM258 for ORF 3, XM1024/XMI023 for ORF 4, PP287/PP286 for ORF 5, PP289/XM780 for ORF 6, andPP285/PP284 for ORF 7 (Table 4).

[0206] Cloning, sequencing and nucleotide sequence analyses. Primers forRT-PCR were designed on the basis of PRRSV isolate VR 2385 sequences,which amplified the entire protein coding regions of ORFs 2 to 5 ofPRRSV isolates ISU 79 and ISU 1894. Primers JM259 and JM260 were usedfor amplification of ORFS 4 and 5, and XM992 and XM993 for amplificationof ORFs 2 and 3 (Table 4). Unique restriction sites (EcoRi and BamHI) atthe termini of the PCR products were introduced, thus enabling acassette approach to replacement of these ORFS.

[0207] The PCR products of ORFs 2-3 and ORFs 4-5 of ISU 79 and ISU 1984were each digested with EcoRi and BamiHI, then purified and cloned intovector pSK+ as described previously (Meng et al., 1993, J. Vet. Diagn.Invest., 5:254-258). Plasmids containing viral inserts were sequencedwith a conventional automated DNA sequencer (Applied Biosystem, Inc.).At least three cDNA clones representing the entire sequence of ORFs 2 to5 from each virus isolate were sequenced with universal and reverseprimers, as well as other virus-specific sequencing primers (XM969,XM970, XM1006, XM078 and XM077; see Table 4).

[0208] To determine the leader-mRNA junction sequences of sg mRNAs 3, 4and 4-1, primer pair IM755 and DP586 (Table 4) was used for RT-PCR toamplify the corresponding 5′-terminal sequences. The resulting PCRproducts were purified and sequenced-by direct PCR sequencing usingvirus specific primers XMD77 and XM141 (Table 4). The sequences werecombined and analyzed by MacVector (International Biotechnologies, Inc.)and GeneWorks (IntelliGenetics, Inc.) computer software programs.

[0209] Oligonucleotides.

[0210] The synthetic oligonucleotides used in this study were summarizedin Table 4. These oligonucleotides were synthesized as single strandedDNA using an automated DNA synthesizer (Applied Biosystem) and purifiedby high pressure liquid chromatography (HPLC).

Results

[0211] sg mRNAs are not packaged into PRRSV virions. To determinewhether the sg mRNAs of PRRSV are packaged, virions of PRRSV isolate ISU3927 were purified by CsCl gradient. The purified virions were treatedwith RNase A before pelleting the virion and extracting RNA, to removeany RNA species which may have adhered to the virion surface. RNAs fromRNase A-treated virions along with the total intracellular RNAs fromisolate ISU 3927 of PRRSV-infected cells were separated in aformaldehyde gel and hybridized with a probe generated from the3′-terminal sequence of the viral genome by PCR with primers PP284 andPP285 (U.S. application Ser. No. 08/131,625; Table 4).

[0212] Only the genomic RNA was detected in the purified virions ofPRRSV isolate ISU 3927 (FIG. 4), and no detectable amounts of sg mRNAswere observed in the purified virions even after 3 weeks exposure. Incontrast, seven species of sg mRNAs, in addition to the genomic RNA,were detected in ISU 3927-infected cells (FIG. 4). Similar results wereobserved with two other U.S. isolates, ISU 55 and ISU 79.

[0213] Variation in the numbers of the sg mRNAs among U.S. PRRSVisolates with differing virulence. All arteriviruses known prior to thepresent invention, including U.S. PRRSV and European PRRSV, have beenshown to produce six sg mRNAs, except for three LDV variants (LDV-P,LDV-a and LDV-v), which synthesize seven sg mRNAs. However, a nested setof six sg mRNAs is produced in the LDV-C strain.

[0214] To compare if there are any variations in the sg mRNAs among U.S.PRRSV isolates, confluent monolayers of CRL 11171 cells were infectedwith five different isolates of U.S. PRRSV with differing virulence at am.o.i. of 0.1. Total intracellular RNAs were isolated fromvirus-infected cells at 24 h post-infection. A cDNA fragment wasgenerated from the extreme 3′-end of the viral genome by PCR withprimers PP284 and PP285 (Table 4). The cDNA fragment was labelled with³²P-dCTP by the random primer extension method, and hybridized with thetotal intracellular RNAs (separated on a formaldehyde gel).

[0215] Analyses of the RNAs showed that a nested set of six or more sgmRNAs, in addition to the genomic RNA, was present in cells infectedwith one of the five isolates of U.S. PRRSV with differing virulence(FIG. 5). Similar results were obtained when the total intracellularRNAs were separated on a glyoxal-DMSO agarose gel. PRRSV isolates ISU55, ISU 79 and ISU 3927 produced seven easily distinguishable sg mRNAs,whereas isolates ISU 22 and ISU 1894 produced six sg mRNAs (FIG. 5). TheU.S. PRRSV isolate VR 2385 also produces six sg mRNAs (U.S. applicationSer. No. 08/131,625). An additional species of sg mRNA was locatedbetween sg mRNAs 3 and 4, and was designated as sg mRNA 4-1. The sgmRNAs differed little, if any, in size among the five isolates of PRRSV(FIG. 5). There appears to be no correlation, however, between thepneumovirulence and the numbers of the sg mRNAs observed in these fiveisolates.

[0216] sg mRNA 4-1 is not a defective-interfering RNA and is not aresult of nonspecific binding of the probes to ribosomal RNAs. It hasbeen shown that, in coronaviruses, a variety of defective interferingRNA (DI RNA) of different sizes were generated when MHV was seriallypassaged in tissue culture at a high m.o.i. DI RNAs were also observedin cells infected with torovirus during undiluted passage. Therefore,the possibility of sg mRNA 4-1 of PRRSV being a DI RNA was investigated.

[0217] To exclude this possibility, the original virus stock of PRRSVisolate ISU 79, which produces the additional species of sg mRNA 4-1,was passaged four times in CRL 11171 cells at different m.o.i. of 0.1,0.01 and 0.001, respectively. In a control experiment, four undilutedpassages of the original virus stock of ISU 79 were performed. Afterfour passages, total intracellular RNAs were isolated fromvirus-infected cells and Northern blot analysis was repeated with thesame probe generated from the extreme 31 -end of the viral genome.

[0218] Analyses of the sg mRNAs showed that the additional species of sgmRNA 4-1 was still present in all RNA preparations with differentm.o.i., as well as in RNA preparations from undiluted passages (FIG.6A). Moreover, there was no interference or reduction in the synthesisof other sg mRNAs in the presence of sg mRNA 4-1, as is usually the casewith DI RNA.

[0219] It has been demonstrated that the DI RNAs of MHV disappearedafter two high-dilution passages. Therefore, if the original virus stockof ISU 79 contained DI RNA, then the DI RNA should disappear after fourhigh-dilution passages. The experimental data above suggests that,unlike DI RNA, the replication of sg mRNA 4-1 is independent of theamount of standard virus. Thus, sg niRNA 4-1 is not a DI RNA.

[0220] In Northern blot analysis of total intracellular RNAs, the probesmay nonspecifically bind to the 18S and 28S ribosomal RNAs, which areabundant in total cytoplasmic RNA preparations. Alternatively, theabundant ribosomal RNAs may cause retardation of virus-specific sg mRNAswhich may co-migrate with the ribosomal RNAs in the gel.

[0221] Two additional bands due to the nonspecific binding of probes tothe ribosomal RNAs have been observed in LV-infected cells andLDV-infected cells. Therefore, it is possible that sg mRNA 4-1 of PRRSVis due to the nonspecific binding of probes to the ribosomal RNAs.

[0222] To rule out this possibility, polyadenylated RNA was isolatedfrom total intracellular RNAs of CRL 11171 cells infected with either oftwo PRRSV isolates, ISU 55 and ISU 79. Both ISU 55 and ISU 79 producethe additional species of sg mRNA 4-1 (FIG. 5). Northern blot analysisof the polyadenylated RNA showed that the additional species of sg mRNA4-1 in cells infected with either of these two isolates was stillpresent (FIG. 6B), indicating that sg mRNA 4-1 is not due to thenonspecific binding of a probe to the ribosomal RNAs.

[0223] The sg mRNAs represent a 3′-coterminal nested set and the sg mRNA4-1 is derived from the sequence upstream of ORF 4. Six sg mRNAs, inaddition to the genomic RNA, are detected in cells infected with VR 2385using a cDNA probe from the extreme 3′-end of the viral genome (U.S.application Ser. No. 08/131,625). Thus, like Berne virus (BEV), LDV,EAV, coronaviruses and LV, the replication of U.S. PRRSV also requiresthe synthesis of a 3′-coterminal nested set of sg mRNAs (U.S.application Ser. Nos. 08/131,625 and 08/301,435).

[0224] To analyze these sg mRNAs in more detail, seven cDNA fragmentsspecific for each of ORFs 1b through 7 were amplified by PCR. The designof primers for PCR was based on the sequence of VR 2385. The sequencesand locations of the primers, IM729 and IM782 for ORF 1b, IM312 andIM313 for ORF 2, XM1022 and IM258 for ORF 3, XM1024 and XM1023 for ORF4, PP286 and PP287 for ORF 5, PP289 and XM780 for ORF 6, and PP284 andPP285 for ORF 7 and the 3′ noncoding region (NCR), are shown in Table 4.The primers were designed in such a way that each set of primers willonly amplify a fragment from a particular ORF, and the overlappingsequences between neighboring ORFs are not included in any givenfragment. Therefore, each of these seven DNA fragments represents onlyone particular ORF except for fragment 7, which represents both ORF 7and the 3′-NCR.

[0225] These seven DNA fragments were labeled with ³²P-dCTP andhybridized to Northern blots of total intracellular RNAs extracted fromcells infected with either of two U.S. isolates of PRRSV, ISU 1894 andISU 79. Total intracellular RNAs isolated from mock-infected CRL 11171cells were included as a control.

[0226] Northern blot analyses showed that Probe 1, generated from ORF1b, hybridized only with the genomic RNA. Probes 2 through 7 eachhybridized with one more additional RNA species besides the genomic RNA(FIG. 7). The results indicate that a 3′-coterminal nested set of six(ISU 1894) or more (ISU 79) sg mRNAs is formed in PRRSV-infected cells(FIGS. 7A and 7B), with the smallest 3′-terminal RNA (sg mRNA 7)encoding ORF 7. The sg mRNAs of U.S. PRRSV all contain the 3′-end of thegenomic RNA, but extend for various distances towards the 5′-end of thegenome, depending on the size of the given sg mRNA.

[0227] The sg mRNA 4-1 of PRRSV isolate ISU 79 hybridized with probes 4through 7, but not with probes 1, 2 and 3 (FIG. 7B), suggesting that sgmRNA 4-1 contains ORFs 4 through 7 as well as the 3′-NCR. Therefore, sgmRNA 4-1 is generated from the sequence upstream of ORF 4.

[0228] A single nucleotide substitution leads to the acquisition of theadditional species of sg mRNA 4-1. Northern blot hybridization datashowed that sg mRNA 4-1 is derived from the sequence upstream of ORF 4(FIG. 7B). To determine the exact location and the leader-mRNA junctionsequence of sg mRNA 4-1, a set of primers, IM755 and DP586, was designed(Table 4). The forward primer IM755 was based on the 3′-end of theleader sequence of VR 2385, and the reverse primer DP586 is located inORF 4 (Table 4).

[0229] RT-PCR with primers IM755 and DP586 was performed using totalintracellular RNAs isolated from cells infected with either of ISU 1894or ISU 79. ISU 79 produces sg mRNA 4-1, but ISU 1894 does not (FIG. 5).A 30-second PCR extension time was applied to preferentially amplify theshort fragments representing the 51-terminal sequences of sg mRNAs 3, 4and 4-1.

[0230] Analysis of the RT-PCR products showed that two fragments withsizes of about 1.1 kb and 0.45 kb were amplified from the total RNAs ofISU 1894 virus-infected cells (FIG. 8A). These two fragments represent5′-portions of sg mRNAs 3 and 4, respectively. In addition to the twofragments observed in the isolate of ISU 1894, a third fragment of about0.6 kb representing the 5′-portion of sg mRNA 4-1 was also amplifiedfrom total RNAs of cells infected with ISU 79 (FIG. 8A).

[0231] To determine the leader-mRNA junction sequences of sg mRNAs 3, 4and 4-1, the RT-PCR products of ISU 79 and ISU 1894 were purified froman agarose gel using a GENECLEAN kit (Bio 101, Inc.), and sequenceddirectly with an automated DNA Sequencer (Applied Biosystems). Theprimers used for sequencing the 5′-end of the RT-PCR products (XM141 andXM077, Table 4) were designed on the basis of the genomic sequences ofISU 79-and ISU 1894 (FIG. 9). The leader-niRNA junction sequences (inwhich the leader joins the mRNA body during the synthesis of sg mRNAs)of sg mRNAs 3, 4, and 4-1 of the two U.S. PRRSV isolates were determinedby comparing the sequences of the 5′-end of the sg mRNAs and the genomicRNA of the two isolates (FIG. 8B).

[0232] The leader-mRNA junction sequences of sg mRNAs 3 and 4 of ISU1894 and ISU 79 were identical. For sg mRNA 3, the leader-junctionsequence (GUAACC) is located 89 nucleotides upstream of ORF 3. For sgmRNA 4, UUCACC is located 10 nucleotides upstream of ORF 4 (FIG. 8B andFIG. 9). The leader-mRNA junction sequence of sg mRNA 4-1 of ISU 79 isUUGACC, located 236 nucleotides upstream of ORF 4 (FIGS. 8B and 9).

[0233] Sequence alignment of the genomic sequences of ISU 79 and ISU1894 shows that a single nucleotide substitution, from T in ISU 1894 toC in ISU 79, leads to the acquisition of an additional leader-mRNAjunction sequence, UUGACC, in ISU 79 (FIGS. 8B and 9). Therefore, anadditional species of sg mRNA (4-1) is formed (FIG. 5). In addition toORFs 4 to 7 contained within sg mRNA 4, sg mRNA 4-1 contains at the51-end an additional small ORF (ORF 4-1) with a coding capacity of 45amino acids (FIG. 9). This small ORF stops just one nucleotide beforethe start codon of ORF 4.

[0234] Sequence analyses of ORPs 2 to 7 of two U.S. isolates revealheterogeneity of the leader mRNA junction sequences. ORFs 2 to 5 of ISU79 and ISU 1894 were cloned and sequenced (see Experiment 1 above). ISU79 produces seven easily distinguishable sg mRNAs, whereas ISU 1894produces six distinguishable sg mRNAs (FIGS. 5 and 7). At least threecDNA clones at any given region of ORFs 2 to 5 were sequenced for eachvirus isolate, using universal and reverse primers as well asvirus-specific primers XM969, XM970, XMI006, XM078, and XM077 (Table 4).The sequences of ORFs 6 and 7 of ISU 1894 and ISU 79 are disclosed inU.S. application Ser. No. 08/301,435.

[0235] Sequence analysis showed that the ORFs 2 to 7 of ISU 79 and ISU1894 overlap each other except for a 10-nucleotide noncoding regionbetween ORF 4 and ORF 5. The same observation was previously made for VR2385 (U.S. application Ser. No. 08/301,435). This is very unusual, sinceall members of the proposed Arteriviridae family, including LV, containoverlapping ORFS. However, the ORFs of coronaviruses are separated byintergenic noncoding sequences. Therefore, U.S. PRRSV appears to besomewhat similar to the coronaviruses in terms of the genomicorganization in junction regions of ORFs 4 and 5.

[0236] ORF 2 of ISU 1894 was one amino acid longer than that of ISU 79(FIG. 9). The stop codon of ORF 2, TAG, was changed to TGG in ISU 1894immediately followed by a new stop codon (TGA) in ISU 1894 (FIG. 9). Thesizes of other ORFs of ISU 79 and ISU 1894 were identical (FIG. 9).There were no deletions or insertions in ORFs 2 to 7 of these isolates.However, numerous substitutions are present throughout the entiresequence of ORFs 2 to 7 between ISU 79 and ISU 1894 (FIG. 9).

[0237] The numbers and locations of the determined or predictedleader-mRNA junction sequences varied between ISU 1894 and ISU 79 (FIG.9). In addition to the regular leader-mRNA 4 junction sequence, TTCACC,10 nucleotides upstream of ORF 4, there was an additional leader-mRNA4-1 junction sequence (TTGACC) located 236 nucleotides upstream of ORF 4in ISU 79 (FIG. 9). The leader-mRNA junction sequences of sg mRNAs 4 and4-1 were separated by 226 nucleotides, which correlated with theestimated sizes of sg mRNAs 4 and 4-1 observed in Northern blot analysis(FIG. 5) and RT-PCR amplification (FIG. 8A).

[0238] The leader-mRNA 3 junction sequence is identical between ISU 1894and ISU 79, GTAACC, located 89 nucleotides upstream of ORF 3. Thepredicted leader-mRNA junction sequences of sg mRNAs 2 and 6 of ISU 1894and ISU 79 were also the same (FIG. 9).

[0239] However, the predicted leader-mRNA 5 junction sequences of ISU1894 and ISU 79 are different (FIG. 9). There are 3 potentialleader-mRNA 5 junction sequences for ISU 79 (GCAACC, GAGACC and TCGACC,located 55, 70 and 105 nucleotides upstream of ORF 5, respectively). Twopotential leader-mRNA 5 junction sequences were also found in ISU 1894(GAAACC and TCGACC, located 70 and 105 nucleotides upstream of ORF 5,respectively) (FIG. 9). The differences were due to the two-nucleotidesubstitutions in the predicted leader-mRNA 5 junction sequences of theseisolates (FIG. 9).

[0240] In addition to the leader-mRNA 7 junction sequence 15 nucleotidesupstream of ORF 7, an additional leader-mRNA 7 junction sequence wasround (ATAACC), located 129 nucleotides upstream of ORF 7 in each ofthese two isolates (FIG. 9). However, the sg mRNA corresponding to thisadditional leader-mRNA 7 junction sequence was not clearlydistinguishable from the abundant sg mRNA 7 which produced awidely-diffused band in the Northern blot (FIGS. 5, 6 and 7).

[0241] Variations in the numbers and locations of the leader-mRNAjunction sequences between LV and the two U.S. isolates analyzed in thisexperiment were also found by comparing the leader-mRNA junctionsequences or LV with those of the two U. S. isolates ISU 1894 and ISU79. Taken together, these data indicate that the sg mRNAs of PRRSV arepolymorphic, and the numbers and the exact sizes of the sg niRNAs dependon the particular PRRSV isolate analyzed. However, a nested set of sixsg mRNAs most likely reflects the standard arterivirus genomeorganization and transcription. TABLE 4 Synthetic oligonucleotides usedin Experiment 2 Oligo Name Sequence Location (nucleotides)^(a)Polarity^(b) IM729 5′-GACTGATGGTCTGGAAAG-3′ (SEQ ID NO:78) ORFlb, −507to −490 upstream of + ORF2 IM782 5′-CTGTATCCGATTCAAACC-3′ (SEQ ID NO:79)ORFlb, −180 to −163 upstream of − ORF2 IM312 5′-AGGTTGGCTGGTGGTCTT-3′(SEQ ID NO:80) ORF2, 131-148 downstream of ORF2 + IM3135′-TCGCTCACTACCTGTTTC-3′ (SEQ ID NO:81) ORF2, 381-398 downstream of ORF2− XM1022 5′-TGTGCCCGCCTTGCCTCA-3′ (SEQ ID NO:82) ORF3, 168-175downstream of ORF3 + IM268 5′-AAACCAATTGCCCCCGTC-3′ (SEQ ID NO:83) ORF3,520-537 downstream of ORF3 − XM1024 5′-TATATCACTGTCACAGCC-3′ (SEQ IDNO:84) ORF4, 232-249 downstream of ORF4 + XM10235′-CAAATTGCCAACAGAATG-3′ (SEQ ID NO:85) ORF4, 519-536 downstream of ORF4− PP287 5′-CAACTTGACGCTATGTGAGC-3′ (SEQ ID NO:86) ORE5, 129-148downstream of ORF5 + PP286 5′-GCCGCGGAACCATCAAGCAC-3′ (SEQ ID NO:87)ORF5, 538-667 downstream of ORF5 − PP289 5′-GACTGCTAGGGCTTCTGCAC-3′ (SEQID NO:88) ORF6, 119-138 downstream of ORF6 + XM7805′-CGTTGACCGTAGTGGAGC-3′ (SEQ ID NO:89) OR66, 416-433 downstream of ORF6− PP285 5′-CCCCATTTCCCTCTAGCGACTG-3′ (SEQ ID NO:90) ORF7, 157-178downstream of ORF7 + PP284 5′-CGGTCGTGTGGTTCTCGCCAAT-3′ (SEQ ID NO:91)31 NCR, −27 to −6 upstream of poly − (A) JM2605′-GGGGAATTCGGGATAGGGAATGTG-3′ (SEQ ID NO:69) ORF3, 338-356 downstreamof ORF3 + JM259 5′-GGGGATCCTTTTGTGGAGCCGT-3′ (SEQ ID NO:68) ORF6, 34-52downstream of ORF6 − XM993 5′-GGTGAATTCGTTTTATTTCCCTCCGGGC-3′ (SEQ IDNO:72) ORFlb, −53 to −35 upstream of ORF2 + XM9925′-GGGGGATCCTGTTGGTAATAG/AGTCTG-3′ (SEQ ID NO:70-71) ORF3, −50 to −34upstream of ORF4 − XM970 5′-GGTTTCACCTAGAATGGC-3′ (SEQ ID NO:74) ORF2,522-550 downstream of ORF2 + XM969 5′-GATAGAGTCTGCCCTTAG-3′ (SEQ IDN0:73) ORF5, 443-460 downstream of ORF6 − XM1006 5′-GCTTCTGAGATGAGTGA-3′(SEQ ID N0:75) ORF4, 316-332 downstream of ORF4 + XM0785′-CTGAGCAATTACAGAAG-3′ (SEQ II) NO:76) ORF2, 202-218 downstream ofORF2 + XM077 5′-CAACCAGGCGTAAACACT-3′ (SEQ ID NO:95) ORF3, 316-333downstream of ORF3 − IM755 5′-GACTGCTTTACGGTCTCTC-3′ (SEQ ID NO:92)Leader, 3′`end of the leader + sequence DP586 5′-GATGCCTGACACATTGCC-3′(SEQ ID NO:93) ORF4, 355-372 downstream of ORF4 − XM1415′-CTGCAAGACTCGAACTGAA-3′ (SEQ ID NO:94) ORF4, 78-97 downstream of ORF4−

EXAMPLE 3

[0242] Cell line ATCC CRL 11171 was used for the propagation of PRRSVisolates. The maintenance of the cell line and isolation of virus werethe same as previously described (Meng et al., J. Gen. Virol.75:1795-1801 (1994); Meng et al., J. of Veterinary DiagnosticInvestigation 8:374-381 (1996)). Plasmacytoma cell line SP2/0 was usedfor cell fusion in MAb preparation. PRRSV ATCC VR 2385 was used asantigen for screening of hybridomas secreting PRRSV specific monoclonalantibodies.

[0243] Indirect Immunofluorescence Assay (IFA).

[0244] Monolayers of ATCC CRL 11171 cells were inoculated with PRRSV VR2385 at 0.1 multiplicities of infection, incubated for 48 hrs and fixedwith methanol. Hybridoma supernatant was incubated on the fixed-cellmonolayer at 370C for 30 min. Fluorescein-labeled goat anti-mouse IgG(H+L) conjugate was used to detect the specific reaction. One PRRSV N(ORF 7 products) specific monoclonal antibody, PP7eF11 was used as apositive control and cell culture supernatant from a non-PRRSV specificMAb, PPAc8 was used as a negative control.

[0245] MAb Preparation.

[0246] The whole cell lysates from insect cells infected withrecombinant baculoviruses of PRRSV ORFs 4 and 5 were used as immunogento immunize mice. Construction of the recombinant baculovirusescontaining the PRRSV ORFs 4 and 5 was done with the strategies aspreviously described (Bream et al. J. Virol. 67:2665-2663 (1993)).Briefly, PRRSV ORFs 4 and 5 genes were PCR amplified separately from thetemplate of pPSP.PRRSV2-7 plasmid (Morozov et al., Archives of Virology140:1313-1319 (1995)) with primers containing restriction sites of BamHIand EcoRI. The amplified fragments were cut with the restriction enzymesindicated above and ligated into the vector PVL1393 (Invitrogen). Theinserted genes were under control of the polyhedrin gene promotor(O'Reilly et al., Baculovirus Expression Vectors. A Laboratory Manual,pages 107-234, ₂nd Edition, New York: W.H. Freeman and Company (1992))and verified with restriction enzyme digestion and PCR amplification.Then the recombinant vector DNA and linearized Autographa Californiamultinuclear polyhedrosis virus DNA (Invitrogen) were co-transfectedinto Sf9 cells as described in the instruction manual. The insertedgenes in the recombinant baculoviruses were verified with hybridizationand PCR amplification (O'Reilly et al., 1992). The recombinant viruseswere used to inoculate insect cells and the cell lysate was used forimmunization of mice. The immunization was carried out with 3 to 5 timesof intraperitoneal injections at two weeks interval. Spleenocytes werehybridized with SP2/0 myeloma cells as previously described (Brown &Ling, “Murine Moncolonal Antibodies,” In Antibodies: a practicalapproach, pp. 81-104, Edited by Catty D. Zoxford, Washing, D.C. IRLPress (1988)). Hybridomas were screened for secreting PRRSV specificantibodies with IFA to detect reaction with PRRSV ATCC VR 2385. Positivehybridomas were selected and cloned three times. Four MAbs weredeveloped to the GP4 and six Mabs to the protein. Mabs were isotypedwith MonoAb ID kits (Zymed Laboratories Inc).

[0247] Enzyme-Linked Immunosorbent Assay (ELISA).

[0248] ELISA has been well described (Harlow & Lane, Antibodies: Alaboratory manual, pp. 471-612, Cold Spring Harbor Laboratory New York(1988); Ausubel et al., Short protocols in molecular biology, pp.11.5-11.7, ₂nd Edition, New York, Greene Publishing Associates and JohnWiley & Sons (1992)). Coating antigens were extracted with 1% TritonX-100 from PRRSV VR 2385-infected cells. MAbs were tested for bindingactivity in ELISA with the antigens binding to plates. Extract fromnormal cells and cell culture medium from the non-PRRSV specific MAb,PPAc8 were included as a negative antigen and a negative antibodycontrols respectively. The PRRSV N-specific MAb, PP7eF11 was used as apositive control. Specific reactions were detected with goat anti-mouseIgG (H+L) peroxidase conjugate and revealed with substrate2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)(ABTS). Then theoptical density was measured at 405 nm (A₄₀₅).

[0249] Fixed-cell ELISA was conducted as previously reported (vanNieuwstadt et al., J. Virol. 70:4767-4772 (1991)) to test the reactivityof MAbs with PRRSV field isolates. Briefly, monolayers of ATCC CRL 11171cells were inoculated with PRRSV field isolates at 0.001 multiplicitiesof infection, incubated for 48 hrs and fixed with methanol. Then thecells were blocked with 1% BSA for 1 hour at room temperature. Cellculture supernatant of MAbs were diluted in two-fold series and added tothe fixed-cell plates. The PP7eF11 and PPAc8 were used as positive andnegative controls respectively. Specific reactions were detected asdescribed above.

[0250] Immunoblotting.

[0251] Western immunoblot analyses were carried out as describedpreviously (Harlow & Lane, Antibodies:A laboratory manual, pp. 471-612,Cold Spring Harbor Laboratory, New York (1988)). Protein samples weretreated under different conditions before separated in gel. Fordenaturing conditions samples were treated at 100° C. for 3 minutes inLaemmli sample buffer containing 2% SDS and 5% 2-mercaptoethanol and runin SDS-PAGE. Under non-denaturing conditions, samples were treated at40° C. for 20 min in sample buffer containing 1% triton X-100 and run inPAGE. Then separated proteins were transferred to nitrocellulosemembrane by electrophoresis. The nitrocellulose membrane was blockedwith 3% BSA. MAbs were screened for the reactivity with the antigens onthe membrane with multi-screening apparatus. Pig anti-PRRSV serum wasused as a positive control and cell culture supernatant from PPAc8 as anegative control. Bound antibodies were detected by in cubation withgoat anti-mouse IgG+IgA+IgN peroxidase conjugate or goat anti-pig IgGperoxidase conjugate followed by color development in4-chloro-1-naphthol substrate.

[0252] Virus Neutralization (VN) Test.

[0253] Virus neutralizing activity of MAbs was tested as describedpreviously (Mecham et al., Viral Immunol. 3:161-170 (1990) & White etal., J. Gen. Virol. 71:4767-4772 (1990)) with some modifications.Hybridoma supernatant was mixed with the same volume of PRRSV dilutioncontaining 30-70 plaque forming units, which was diluted with DMEMcontaining 10% guinea pig complement. The virus-antibody mixture wasincubated at 37° C. for 1 hr, and then transferred to the monolayer ofATCC CRL 11171 cells in six-well plate for 1 more hr incubation at 37°C. Then an agarose-medium mixture overlaid the monolayer. After 3-dayincubation at 37° C., the monolayer was stained with 0.05% neutral redin agarose. Pig anti-PRRSV serum was used as a positive control andhybridoma cell culture medium from a non-PRRSV specific MAb was includedas a negative control.

[0254] PRRSV Specific Mabs Identified with IPA.

[0255] Hybridomas were screened with IFA on PRRSV VR 2385-infected ATCCCRL 11171 cells. IFA positive hybridomas were selected, amplified andcloned. Six MAbs were developed against PRRSV E protein and four to theGP4. All of them showed strong perinuclear fluorescence with a littledifference in intensity, which was different from the cytoplasmicstaining of PRRSV N protein specific MAb (FIG. 10). This resultindicated that the GP4 and E glycoproteins were synthesized andaccumulated in subcellular compartments in PRRSV-infected cells astransferring of oligosaccharides to a glycoprotein is generallyprocessed in a particular compartment such as the endoplasmic reticulumand the Golgi complex (Pfeffer et al., Ann. Rev. Biochem. 56:829-852(1987)). GP4 and E were predicted as membrane-associated glycoproteins(Meng et al., 1994 & Morozov et al., Archives of Virology 140:1313-1319(1995)). In contrast, the PRRSV N protein is highly basic andhydrophilic, and is synthesized in the cytoplasm of PRRSV-infectedcells, which was shown by the observation of cytosol distribution offluorescence in IFA with N-specific MAb staining. All the MAbs wereidentified as subtype IgM.

[0256] Reactivity with PRRSV Antigen in ELISA.

[0257] In order to determine the sensitivity of the epitopes todetergent treatment, ELISAs were run to test the reactivity of the MAbswith 1% Triton X-100 extracted PRRSV antigen. Among the MAbs to the Eprotein, only PP5bH4 showed strong reactivity to the PRRSV antigen (FIG.11). No clear reaction was detected between the rest of the E-specificMAbs and the PRRSV antigen. Among the MAbs to the GP4, only PP4bB3showed a mild reactivity with the PRRSV antigen. The other three of theMAbs to GP4 failed to show any reactivity. The negative controls did notshow reaction in ELISA.

[0258] Out of the 10 MAbs, only PP5bH4 and PP4bB3 showed reactivity inthe ELISA with detergent extracted PRRSV antigen. This result indicatedthat the epitope recognized by PP5bH4 was resistant to Triton X-100treatment and the epitope of PP4bB3 was partially resistant to thedetergent. The epitopes recognized by the other 8 MAbs were sensitive tothe treatment, and may be conformationally dependent. Triton X-100 isgenerally selected to disrupt cell membranes for its nondenaturingproperty (Deutscher, “Guide to protein purification,” Methods inEnzymology, Vol. 182, San Diego, Calif., Academic Press, Inc. (1990)),but in this test the epitopes in the PRRSV proteins were somehow alteredduring the extraction process as monitored by the MAb binding.

[0259] Immunoblotting Assay.

[0260] Western-blotting was carried out to determine the reactivity ofthe MAbs with PRRSV antigen to confirm the speculation that the MAbswere against conformationally dependent epitopes. Under denaturedconditions in SDS-PAGE, only the PP5bH4 recognized a band of purifiedPRRSV virions in the position of 26 kDa which corresponded with theputative E detected with pig anti-PRRSV serum (FIG. 12). Thenimmunoblotting was carried out with non-denatured PAGE to test if theepitopes were preserved under nondenaturing conditions. Among the sixMAbs to E, only PP5bH4 showed reaction with the PRRSV antigen. Of theMAbs to GP4, none recognized the PRRSV antigen in purified virions or ininfected cells under either conditions in this test (result not shown).

[0261] The MAbs except PP5bH4 failed to recognize the PRRSV antigen inimmunoblot, which indicated that the epitopes recognized by these MAbswere not derived from continuous structure. MAb PP5bH4 reacted withPRRSV in the position of 26 kDa, which confirmed the report about themolecular mass of E (Meulenberg et al., Virology 192:62-72 (1995)). Thisresult showed that the epitopes recognized by the other 9 MAbs weresensitive to detergent treatment and corresponded to that of ELISA.Again the result indicated that the epitopes were conformationallydependent. PP4bB3 failed to show any reaction with PRRSV antigen inWestern-blot, which could be due to the epitope loss or alternationduring PAGE-and transfer. The sample of purified PRRSV showed threebands with molecular mass of about 19, 26-31 and 45 kDa (FIG. 12). Themissing 15 kDa band of N protein could not be explained.

[0262] Virus Neutralizing Activity.

[0263] Plaque-reduction assay was run to test whether there was anyvirus neutralizing activity among the MAbs to the E and GP4 proteins.Only one E-specific MAb, PP5dB4 showed the ability of homologousneutralization to the VR 2385 isolate. All the other MAbs failed to showany neutralizing activity to this isolate. The positive control, piganti-PRRSV serum also showed virus neutralizing activity.

[0264] Among the ten MAbs to GP4 and E, at least PP5dB4 showedhomologous virus neutralizing activity against PRRSV VR 2385. Theneutralizing epitope was conformationally dependent as PP5dB4 failed torecognize PRRSV antigen in ELISA and in Western-blot. Also theneutralizing activity of PP5dB4 indicates that at least part of theepitope is located on the virion surface and accessible by the MAb. Themechanism of neutralizing activity of PP5dB4 is not clear. It could bedue to blocking of the virus binding or entry into the cells.

[0265] Reactivity with Other PRRSV Isolates.

[0266] PRRSV field isolates were propagated to test the cross-reactivityof the MAbs in fixed-cell ELISA and to determine the epitope presence inother PRRSV isolates (Table 5). Fixed-cell ELISA was used because mostof these MAbs recognized conformationally dependent epitopes and theseepitopes could be preserved in fixed cells. All the MAbs react with allthe isolates but with different titers. The result indicates that theepitopes recognized by the MAbs were conserved among the isolatestested. However, there were antigenic differences among the isolatestested. Reactivity intensity was arbitrarily defined as high if titerswere greater than or equal to 256, as medium if titers were 64 to 128,and as low if titers were smaller than or equal to 32. Out of the 23isolates tested, only PRRSV VR 2385 had high reactivity with 7 of the 10MAbs. Five isolates had low reactivity with at least 6 of the 10 MAbs,12 isolates had medium reactivity with at least 6 of the 10 MAbs and theother 5 isolates had low reactivity with half of the MAbs. The MAbPP4dG6 and PP5bH4 showed lower reactivity with most of the isolates thanother MAbs. The PP4bB3 showed the strongest reactivity among all theMAbs against GP4 and E proteins. The titer difference was as high as64-fold for the reaction of one MAb with the different isolates, such asthe titers of MAb PP4cB11 reacting with PRRSV RP 10 and RP 12, 16 and1024 respectively. On the other hand, the titer difference of MAbs withone isolate was as high as 128-fold, such as the titers of MAbs PP4bB3and PP4bC5 reacting with PRRSV RP11, 1024 and 8 respectively. Thisresult indicated that the epitopes recognized by the different MAbs weredifferent. The positive MAb control show strong reactivity with all theisolates except the ISU-51. The reactivity difference of MAbs with PRRSVisolates was consistent with the report that the amino acid sequenceidentity of VR 2385, ISU22, ISU55 and RP45 was 94-98% in ORF 4 and88-97% in ORF 5 (Meng et al., J. Gen. Virol. 140:745-755 (1995)).

[0267] In summary, six MAbs were developed to the PRRSV E protein andfour to the GP4. All of them except PP5bH4 were against conformationallydependent epitopes as determined by ELISA and immunoblotting. MAb PP5dB4showed virus neutralizing Activity against VR 2385. Reactivity patternof the MAbs with PRRSV field isolates indicated that there are antigenicdifference in PRRSV GP4 and E, which confirmed previous reports on MAbsagainst PRRSV N and ORF 3 product (Nelson et al., J. ClinicalMicrobiology 31:3184-3189 (1993); Drew et al., J. General Virol.76:1361-1369 (1995); Wieczorek-Krohmer et al., Veterinary Microbiology51:257-266 (1996)).

EXAMPLE 4

[0268] Cells and Viruses.

[0269] ATCC CRL11171 cells were used to propagate PRRSV and PRRSVpurification was done as previously described (Meng et al., J. Gen.Virol., 7S:1795-1801 (1994); Meng et al., J. Vet. Diag. Invest.8:374-381 (1996); Halbur et al. Vet. Pathol. 32:648-660, (1995). PRRSVisolate ATCC VR 2385 (Meng et al., 1994 & Morozov et al., 1995) was usedfor PCR amplification of ORFs 2 to 4 genes.

[0270]Spodoptera frugiperda clone 9 (Sf9) and High Five™ (Invitrogen)insect cells were cultured for propagation of baculovirus. Thebaculovirus strain Autographa California multinuclear polyhedrosis virus(AcMNPV) was used as parent virus for recombinant baculovirusconstruction.

[0271] Construction of AcMNPV recombinant transfer vector. Constructionof the baculovirus transfer vectors containing the PRRSV ORFs 2, 3 and 4separately was done with the strategies as previously described (Breamet al., J. Virol. 67:2655-2663(1993). Briefly, PRRSV ORFs 2 to 4 geneswere PCR amplified separately from the template of pPSP.PRRSV2-7 plasmidwith primers containing restriction sites of BamIII and Pst I for genesof ORFs 2 and 3, BamHI and EcoRI for ORF 4.

[0272] The forward primer for ORF 2 was 5′GCACGGATCCGAATTAACATGAAATGGGGT-3′ (SEQ ID NO:96) and the reverse primer was5°CCACCT GCAGATTCACCGTGAGTTCGAAAG-3′ (SEQ ID NO:97). The forward primerfor ORF 3 was 5′CGTCGGATCCTCCTACAATGGCTAATAGCT-3′ (SEQ ID NO: 105) andthe reverse primer was 5′CGCGCTGCAGTGTCCCTATCGACGTGCGGC-3′ (SEQ ID NO:106). The forward primer for ORF 4 was5′GTATGGATCCGGCAATTGGTTTCACCTATAA-3′ (SEQ ID NO:107) and the reverseprimer was 5′ATAGGAATTCAACAAGACGGCACGATACAC-3′ (SEQ ID NO: 108). Theamplified fragments were cut with restriction enzymes as indicated aboveand ligated into the vector pFastBAC1 (GIBCO BRL) for ORFs 2 and 3fragments, and the vector PVL1393 (Invitrogen) for ORF 4 fragment. Theinserted genes were under control of the polyhedrin gene promotor(O′Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual,W.H. Freeman & Co., NY (1992) and verified with restriction enzymedigestion and PCR amplification. Then the recombinant vectors containingthe ORFs 2 to 4 genes separately were isolated and designated aspPSP.Ac-p2 for ORF 2 transfer vector, pPSP.Ac-p3 for ORF 3 transfervector and pPSP.Ac-p4 for ORF 4 transfer vector. For pPSP.Ac-p2 andpPSP.Ac-p3, their DNA were isolated and transfected into competentDH1OBAC E. coli cells (GIBCO BRL) containing the whole genome ofbaculovirus called Bacmid.

[0273] Transfection and Selection of Recombinant Viruses.

[0274] For ORFs 2 and 3, recombinant viruses were-generated with theBAC-TO-BAC™ expression system (GIBCO BRL) The isolated recombinantBacmid DNA were transfected into Sf9 insect cells and then the cellculture medium was collected as virus stock. For ORF 4 recombinant virusconstruction, pPSP.Ac-p4 DNA and linearized AcMNPV DNA (Invitrogen) wereco-transfected into Sf9 cells as described in the instruction manual.Putative recombinant baculoviruses were selected following three roundsof occlusion body-ndgative plaque purification. The inserted genes inthe recombinant viruses were verified with hybridization and PCRamplification (O'Reilly et al., 1992). Four recombinants were selectedfor each of the 3 strains of recombinant baculoviruses. Indirectimmunofluorescence assays with pig anti-PRRSV serum showed that the fourrecombinants for each strain had similar level of protein expression.One was chosen from each strain for further study and designated asvAc-P2 for recombinant virus of ORF 2, vAc-P3 for that of ORF 3, andvAc-P4 for that of ORF 4.

[0275] Indirect Immunofluorescence Assay (IFA).

[0276] IFA was well described elsewhere (O'Reilly et al., 1992).Briefly, Monolayer of High Five™ cells were infected with wild type (wt)AcMNPV or recombinant viruses of vAc-P2, vAc-P3 and vAc-P4 respectivelyat a multiplicity of infection of 0.1 and incubated for 72 hrs. Piganti-PRRSV serum was used to detect specific proteins expressed ininsect calls. Total protein expression was detected in the infectedcells fixed, stained and observed under fluorescence microscope. Cellsurface expression was detected on unfixed and unpermeabilized cellsincubated with pig anti-PRRSV serum for 1 hr at 4° C., stained withfluorescein-labeled goat anti-pig IgG conjugate for 1 hr at 4° C., andthen observed under fluorescence microscope.

[0277] Immunoblotting.

[0278] Western immunoblotting was conducted as previously described(Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory (1988)). Cell extract from insect cells infected withrecombinant viruses or wt AcMNPV were used for this analysis. Theproteins were separated with SDS-PAGE and transferred to nitrocellulosemembrane by electrophoresis. The membrane was incubated with piganti-PRRSV serum for 1 hour at room temperature. Specific reactions weredetected with goat anti-pig IgG peroxidase conjugate, followed by colordevelopment in 4-chloro-1-naphthol substrate.

[0279] Tunicamycin Treatment.

[0280] High Five™ cells were infected with vAc-P2, vAc-P3, vAc-P4 or wtAcMNPV and incubated with 5 μg/ml tunicamycin in cell-culture mediumfrom 0 to 72 hrs post infection. Non-treated insect cells were infectedat the same time as controls. Cell lysate was harvested for SDS-PAGE andimmunoblotting (O'Reilly et al., 1992).

[0281] Immunogenecity of the Recombinant Proteins.

[0282] Cell lysates of insect cells infected with vAc-P2, vAc-P3 andvAc-P4 were used to test the recombinant protein's immunogenecity inrabbits. Two twelve-week old rabbits were injected intramuscularly andsubcutaneously for each of these recombinant proteins. Blood wascollected 10 days after two booster injections. Antibodies were testedwith indirect ELISA (Ausubel et al., Short Protocols in MolecularBiology, pp. 11.5-11.7, ₂nd Edition, N.Y. Green Publishing Associatesand John Wiley and Sons (1992)). Purified PRRSV virions were sonicatedand used to coat 96-well plates and goat anti-rabbit IgG peroxidaseconjugate was used to detect anti-PRRSV antibodies in rabbit serumsamples. Pre-immune rabbit serum was used as negative control. Substrate2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was usedto reveal specific reactions.

[0283] Results

[0284] Construction and Verification of Recombinant Viruses.

[0285] Details of construction strategy are mentioned under Methods. ForORFs 2 and 3, the recombinant baculoviruses were selected from E. colicontaining the recombinant Bacmid and then collected from transfectionof Sf9 insect cells. The recombinant viruses were further confirmed byDNA hybridization and PCR amplification. Both hybridization of DNA frominfected cells with specific probes from the PRRSV genes of ORFs 2 to 4and PCR amplification showed that the recombinant baculoviruses had theright genes cloned (data not shown).

[0286] Surface Immunofluorescence of Recombinant viruses vAc-P2, vAc-P3and vAc-P4. High Five™ cells were infected with vAc-P2, vAc-P3, vAc-P3,vAc-P4, or wt AcMNPV, incubated for 72 hrs, and fixed with methanol forexamination of total protein expression by IFA with pig anti-PRRSVserum. Unfixed and unpermeabilized insect cells were stained at 4° C. todetect cell surface immunofluorescence by IFA. There was weakcytoplasmic fluorescence in vAc-P2 infected cells, intense cytoplasmicfluorescence in vAc-P3 or vAc-P4 infected insect cells and no specificfluorescence in wt AcMNPV infected cells (FIG. 13). There was clear cellsurface immunofluorescence in vAc-P2, vAc-P3 and vAc-P4 infected insectcells stained at 4° C. without fixation and permeabilization (FIG. 14).No cell surface staining was detected in wt AcMNPV infected insectcells. Also, recombinant virus infected insect cells in the absence ofantibody did not show any fluorescence (data not shown).

[0287] Analysis of Expressed Recombinant Proteins.

[0288] Monolayer of High Five™ cells was infected at a multiplicity ofinfection of 0.1 with vAc-P2, vAc-P3, vAc-P4, or wt AcMNPV and incubatedfor 72 hrs. Expression of the recombinant proteins in insect cells wasanalyzed with whole cell extracts. Total protein samples were run onSDS-PAGE, transferred to nitrocellulose membrane by western-blotting anddetected with pig anti-PRRSV serum. Purified PRRS virions were added andanalyzed in the same gel. The ORF 2 product expressed in insect cellswas detected as 27 and 29 kDa bands in Mr. The ORF 3 product wasdetected as 22, 25, 27-31 and 35-43 kDa multi-band species. The signalsin M_(r) of 27-31 and 35-43 kDa were hard to differentiate into singlebands and may be due to differential glycosylation or partialproteolysis. The ORF 4 product was found as 15, 18, 22, 24, 28 and 30kDa multi-band species. These specific bands were not detected in wtAcMNPV infected insect cells. There were at least four bands in purifiedPRRSV sample: 15, 19, 27-31 and 45 kDa in Mr. The specific bandsdetected in purified PRRS virions were not observed in normal cellcontrol.

[0289] The Recombinant Proteins were Glycosylated.

[0290] Tunicamycin treatment of insect cells infected with recombinantbaculoviruses or wt AcMNPV was conducted to test if the recombinantproteins were N-glycosylated as tunicamycin inhibit N-linkedglycosylation. After the treatment, the 29 kDa band of the ORF 2recombinant protein was disappeared, a 25 kDa appeared and the 27 kDaspecies remained unchanged. For the ORF 3 recombinant protein, thespecies of 27-31 and 35-43 kDa were disappeared and the 22-27 kDa bandsremained unchanged. The 27 kDa species of ORF 3 recombinant proteinbecame more abundant after tunicamycin treatment. After theN-glycosylation inhibition, the ORF 4 recombinant protein was shown as15 and 18 kDa species only and the bands of 22-30 kDa were disappeared.The 15 and 18 kDa bands became sharper and darker after the tunicamycintreatment. No signal was detected in extracts from wt AcMNPV infectedinsect cells.

[0291] Immunogenecity of the Recombinant Proteins.

[0292] The recombinant proteins of ORFs 2 to 4 products were tested forimmunogenecity by immunization of rabbits with lysates of insect cellsinfected with vAc-P2, vAc-P3 and vAc-P4. The presence of anti-PRRSVantibodies in the rabbit serum samples was detected by ELISA. Theaverage titers of immunized rabbits were 192, 128 and 382 for the groupsof vAc-P2, vAc-P3 and vAc-P4 cell lysate respectively (Table 6).

[0293] Discussion

[0294] The genes of ORFs 2 to 4 of PRRSV were cloned into BEVS and therecombinant proteins were expressed in insect cells. The cloningstrategy for ORFs 2 and 3 was much faster than that for ORF 4 as theselection process of recombinant baculovirus was done in E. coli insteadof choosing occlusion body-negative plaques on Sf9 cells. Sf9 cells wereused for the propagation of baculovirus, and High Five™ cells were usedfor protein expression as protein yield in High Five™ cells was believedto be higher than that in Sf9 cells (Wickham et al. BiotechnologyProgress 8:391-396 (1992) & Davis et al., in vitro Cell andDevelopmental Biology 29A: 388-390 (1993)). The High Five™ cells wereadapted to serum free medium, which benefits for future proteinpurification, and can be adapted to suspension culture, which issuitable for large scale industrial production.

[0295] The recombinant proteins were shown by IFA to express in insectcells infected with vAc-P2, vAc-P3 and vAc-P4 recombinant viruses. Therewas weak cytoplasmic fluorescence in vAc-P2 infected cells, strongcytoplasmic fluorescence in vAc-P3 and vAc-P4 infected cells. The reasonfor the weak fluorescence of vAc-P2 infected cells is not known andcould be due to epitope alternation after fixation with methanol. Theunfixed and unpermeabilized insect cells were stained at 4° C. to makesure that the pig anti-PRRSV antibody reacted with cell surface proteinsonly and did not enter into cytoplasm. There was clear cell surfaceimmunofluorescence on the insect cells infected with vAc-P2, vAc-P3 orvAc-P4, which indicates that the recombinant proteins were efficientlyprocessed and transported to cell surface. This result indicates thatORFs 2 to 4 products are membrane-associated proteins, which isconsistent with the predictions from sequence studies (Morozov et al.,Archives of Virology 140:1313-13,19 (1995)). However, it is not clear ifthese products are also transported to cell surface of PRRSV infectedmammalian cells or assembled into virions as surface proteins. Recentreport showed that the ORFs 3 and 4 products are viral structuralproteins (Van Nieuwstadt et al, J. Virol. 70:4767-4772 (1996)). Furtherexperiment is needed to investigate the destiny of these proteins.

[0296] Immunoblotting results showed that the recombinant proteins wereefficiently expressed in insect cells. The ORF 2 product was detected as27 and 29 kDa species in M_(r). Tunicamycin treatment eliminated the 29kDa band and introduced the 25 kDa species with the 27 kDa unchanged,which indicated that the 29 kDa was N-glycosylated. The predicted M, ofPRRSV VR 2385 ORF 2 is 29.5 kDa with two potential glycosylation sites(Morozov et al., 1995). The 25 kDa species may be the core protein ofORF 2 if the 37-38 signal sequence (Meulenberg et al., Virology192:62-72 (1995)) are removed in the mature protein. The 4 kDadifference between the 29 and 25 kDa bands may be due to carbohydratestructures as one glycosyl moiety has a Mr of about 2-3 kDa (Trimble etal., J. Biol. Chem. 250:2562-2567 (1983)). The 27 kDa species was notsensitive to the tunicamycin treatment and may be modified by O-linkedglycosylation or other post-translational modifications.

[0297] The ORF 3 product in insect cells was shown as 22-43 kDamulti-band species detected by immunoblotting. The 28-43 kDa specieswere eliminated by tunicamycin treatment of vAc-P3 infected insectcells, which indicated that they were N-linked glycoproteins and themulti-bands were due to differential glycosylation. The predicted M_(r)of PRRSV VR 2385 ORF 3 product is 28.7 kDa (about 2 kDa less than thecounterpart of IV) with 7 potential N-linked glycosylation sites(Morozov et al., 1995). The 27 kDa species of ORF 3 recombinant proteinmay be the core protein because it became more abundant aftertunicamycin treatment and because a 27 kDa band appeared and a 45 kDaband disappeared after endoglycosidase F treatment of purified PR RSVvirion (data not shown). The species smaller than 27 kDa may betruncated proteins or products of proteolysis. The 27-43 kDa bands innontreated sample are hard to differentiate into individual bands, whichmay be due to overloading or partial proteolysis. The 43 kDa species maybe the fully glycosylated product as there are 7 N-linked glycosylationsites and about 2-3 kDa are counted for each glycosyl moiety (Trimble etal., 1983). The recent report showed that ORF 3 of IV encode a 45-50 kDastructural protein and that recombinant proteins of ORF 3 in insectcells were detected as 28-44 kDa in M, by radioimmunoprecipitation (VanNieuwstadt et al., 1996). The 28 kDa species was found as the coreprotein of LV ORF 3 product. It seems there is a difference in M_(r) ofrecombinant proteins from ORF 3 of US PRRSV and LV, which may be due tothe different expression system used or the difference in this genebetween the two isolates. Another report showed that the recombinantfusion protein of carboxyterminal 199 amino acids of LV ORF 3 expressedin baculovirus was not N-glycosylated (Katz et al., Vet. Microbiol.44:65-76 (1995)), which demonstrates the diversity of expressed productsfrom the same gene.

[0298] The ORF 4 product in insect cells was detected as 15-30 kDamulti-band species. After tunicamycin treatment the 22-30 kDa bands wereeliminated and the 15, 18 kDa bands remained unchanged, which indicatedthat the 22-30 kDa species were N glycosylated to various degrees. TheORF 4 of PRRSV VR 2385 was predicted to encode a 19.5 kDa protein with 4potential N glycosylation sites (Morozov et al., 1995). The 15 kDaspecies of ORF 4 product may be the core protein and the 18 kDa band maybe the core protein plus O-linked glycosyl moiety or othermodifications. It was reported that LV ORF 4 encoded a 31-35 kDastructural protein and that the recombinant protein of ORF 4 expressedin insect cells was detected as 20-29 kDa species with a 17 kDa coreprotein (Van Nieuwstadt et al., 1996). Again, the reason for thedifference in M_(r) may be due to the cloned gene's difference and thedifferent expression systems. Another report demonstrated the differenceby showing that ORF 4 is not a well conserved region (Kwang et al., J.Vet. Diag. Invest. 6:293-296 (1994)).

[0299] The immunization of rabbits with the recombinant proteins showedthat they had induced anti-PRRSV antibodies. This result indicates thatthese recombinant proteins may have the similar immunogenecity as theirnative counterparts in PRRSV infected mammalian cells.

[0300] [a.] This study showed that the ORFs 2 to 4 of PRRSV VR 2385 wereexpressed in BEVS and detected both in cytoplasm and on cell surface ofinsect cells. The recombinant proteins of ORFs 2 to 4 were N-linkedglycoproteins with differential glycosylation. The purified PRRSVvirions were analyzed as the same time and showed 4 bands inimmunoblotting. But due to lack of oligoclonal or monoclonal antibodiesit is hard to tell if any of ORFs 2 to 4 products was detected in thepurified virions. The reaction of pig anti-PRRSV serum with therecombinant proteins indicated that the native counterpart of theseproteins induced immune response in natural host. The induction ofanti-PRRSV antibodies in rabbits indicated that these recombinantproteins had similar immunogenecity as the native ORFs 2 to 4 productsin PRRSV infected natural host. TABLE 6 Rabbit antiserum titers testedwith ELISA Groups of insect cells infected with Number of rabbits Meansof titers* vAc-P2 2 192 vAc-P3 2 128 vAc-P4 2 384

EXAMPLE 5

[0301] Cells and Viruses.

[0302] ATCC CRL1 1171 cells were used to propagate PRRSV (Meng et al.,1994 and 1996; Halbur et al., 1995). Spodoptera frugiperda clone 9 (Sf9)and High Five™ (Invitrogen) insect cells were used for propagation ofbaculovirus. PRRSV isolate VR 2385 (Meng et al., 1994 and 1996) was usedfor gene amplification and cloning into BEVS. PRRSV virions werepurified as previously described (Meng et al., 1994). The baculovirusstrain Autographa california multinuclear polyhedrosis virus (AcMNPV)was used as parent virus for recombinant virus construction.

[0303] Construction of ACMNPV Recombinant Transfer Vector.

[0304] The nucleic acid sequence of the ORFs 5-7 of PRRSV VR2385 waspreviously described (Meng et al. 1994). Construction of the baculovirustransfer vectors containing the PRRSV ORFs 5 to 7 separately was donewith the strategies as described previously (Bream et al. 1993).Briefly, PRRSV ORFs 5 to 7 genes were PCR amplified separately from thetemplate pPSP.PRRSV2-7 plasmid with primers containing restriction sitesof BamHI and EcoRI. The forward primer for ORF5 was5′TGCCAGGATCCGTGTTTAAATATGTTGGGG-3′ (SEQ ID NO:98) and the reverseprimer was 5′CGTGGAATTCATAGAAAACGCCAAGAGCAC-3′ (SEQ ID NO:99). Theforward primer for ORF6 was 5′GGGGATCCAGAGTTTCAGCGG-3′ (SEQ ID NO:100)and the reverse primer was 5′GGGAATTCTGGCACAGCTGATTGAC-3′ (SEQ IDNO:101). The forward primer for ORF7 was 5′GGGGATCCTTGTTAAATATGCC-3′(SEQ ID NO:102) and the reverse primer was 5′GGGAATTCACCAGCATTC-3′ (SEQID NO:103). The fragments amplified were cut with BamHI and EcoRI,isolated and ligated into vector PVL1393 (Invitrogen) which was also cutwith BamHI and EcoRI to insure correct orientations. The inserted geneswere under control of the polyhedrin gene promotor (O'Reilly et al.,1992) and verified with restriction enzyme digestion and PCRamplification. The recombinant vectors containing the ORFs 5 to 7 genesseparately were isolated, pPSP.Ac-E for ORF5, pPSP.Ac-M for ORF6 andpPSP.Ac-N for ORF7 transfer vectors.

[0305] Transfection and Selection of Recombinant Viruses.

[0306] Sf9 insect cells were cotransfected with linearized AcMNPV DNA(Invitrogen) and recombinant plasmid DNA of pPSP.Ac-E, pPSP.Ac-M, andpPSP.Ac-N respectively as per manufacturer's instructions. Putativerecombinant viruses were selected following three-round of purificationof occlusion-negative plaques. The inserted genes in the recombinantviruses were verified with hybridization and PCR amplification (O'Reillyet al., 1992). Four recombinants were selected for each of the 3 strainsof recombinant viruses and were found to be similar inimmunofluorescence assays using pig anti-PRRSV serum. One recombinantvirus was chosen arbitrarily from each strain and designated as vAc-E1for recombinant virus containing ORF5, vAc-M 1 for that with ORF6, andvAc-N1 for that with ORF7.

[0307] Immunoblotting.

[0308] Western inununoblot analyses were carried out as describedpreviously (Harlow and Lane, 1988). Whole proteins from infected insectcells, purified PRRSV or normal cells were used as samples. Proteinswere separated with SDS-PAGE and transferred to nitrocellulose membraneby electrophoresis. The nitrocellulose membrane was blocked with 3% BSAand reacted with pig anti-PRRSV serum for 1 hour at room temperature.Bound antibodies were detected by incubation with goat anti-pig IgGperoxidase conjugate, followed by color development with4-chloro-l-naphthol substrate.

[0309] Tunicamycin Treatment.

[0310] Infected High Five™ cells were incubated with 5 μg/ml tunicamycinin cell-culture medium from 0 to 72 hr post infection and harvested forSDS-PAGE (O'Reilly et al., 1992).

[0311] Cleavage with Glycosidases.

[0312] Endoglycosidase F/N-glycosidase F mixture (PNGase F) andendoglycosidase H (Boehringer-Mannheim Biochemicals) were used to treatlysates from infected High Five™ cells (0.1 PFU/cell; 72 hr postinfection) in the case of recombinant proteins or purified PRRSV as permanufacturer's instructions. Briefly, 10⁵ cells were laced with 30 μglysis buffer. Then 10 μg of cell lysates was digested with PNGase F,endoglycosidase H or kept untreated and used as non-treated control. Thesamples were incubated at 37° C. for 24 hrs before analysis on SDS-PAGE.

[0313] Radioimmunoprecipitation (RIP).

[0314] High Five™ cells infected with recombinant baculovirus or wildtype (wt) AcMNPV and uninfected High Five™ cells were washed once withmethionine-free medium and starved for one hour at 48 hr post-infection.Then 50 μg/ml Tran 35S-label (methionine and cystine) (Amersham LifeScience Inc.) in methionine-free medium was added to the infected cells.Three hours later the cells were rinsed with PBS and laced in RIPA lysisbuffer (10 mM Tris-HCI, pH8.0; 1 mM EDTA; 150 mM NaCl; 1% NP40; 1%sodium deoxycholate; 0.1% SDS). Immunoprecipitation and gelelectrophoresis were performed as described previously (Hutchinson etal., J. Virol. 66:2240-2250 (1992).

[0315] Indirect Immunofluorescence Assay (IFA).

[0316] IFA was conducted as previously described (O'Reilly et al.,1992). Monolayer of High Five™ cells were inoculated with wt AcMNPV orrecombinant baculoviruses, incubated for 72 hrs and fixed to detect allrecombinant protein expression with pig anti-PRRSV serum. The inoculatedinsect cells were also examined for the presence of cell surfaceproteins. Unfixed and unpermeabilized cells were reacted with the pigantiserum at 4° C. for 1 hr, incubated with fluorescein-labeled goatanti-pig IgG conjugate for 1 more hr at 4° C. and then observed underfluorescent microscope.

[0317] Immunogenecity of the Recombinant Proteins.

[0318] Twelve-week old rabbits were injected intramuscularly andsubcutaneously with lysates of insect cells infected with vAc-E1, vAc-M1and vAc-N1. Two rabbits were immunized for each of E, M, and Nrecombinant proteins. Two booster injections were given in an intervalof three weeks. The injection dose was cell lysates from 2×10⁶ insectcells. Blood was collected 10 days after the second booster injection.Antibodies were tested with indirect ELISA. Purified PRRSV virions weresonicated and used to coat 96-well plates and goat anti-rabbit IgGperoxidase conjugate was used to detect anti-PRRSV antibodies inrabbit-serum samples. Pre-immune rabbit serum was used as negativecontrol. Substrate 2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid)(ABTS) was used to reveal specific reactions.

[0319] Results

[0320] Confirmation for the Presence of PRRSV Gene in RecombinantBaculovirus.

[0321] Hybridization and PCR amplification were performed to verify thepresence the cloned genes in recombinant baculovirus. Hybridization ofprobes from the PRRSV genes with recombinant baculovirus showed that thePRRSV genes were present in the recombinant baculovirus. PCRamplification with specific primers from PRRSV genes showed single bandfrom the recombinant virus and absent from the wt AcMNPV (results notshown). These tests confirmed that the recombinant baculoviruses containthe PRRSV genes ORFs 5 to 7. Surface immunofluorescence of recombinantviruses vAc-E1 and vAc-M1, but not vAc-N1. High Five™ cells infectedwith vAc-E1, vAc-M1, vAc-N1, and wt AcMNPV were examined for thepresence of total expressed protein and cell surface expression. Therewas weak cytoplasmic fluorescence in vAc-E1 and vAc-M1-infected cells.In contrast,, there was intense cytoplasmic fluorescence invAc-N1-infected insect cells and no fluorescence in wt AcMNPV infectedcells. Clear cell surface immunofluorescence was detected in vAc-E1 andvAc-M1 infected insect cells. However, there was no surfaceimmunofluorescence in insect cells infected with vAc-N1 or wt AcMNPV.Also, in the absence of antibody insect cells infected with therecombinant viruses did not show any fluorescence (data not shown).

[0322] Analysis of ORFs 5-7 Products Expressed in Insect Cells.

[0323] To analyze the expression of the expected proteins in insectcells, confluent monolayers of High Five™ cells were infected at amultiplicity of infection of 0.1 PFU/cell with vAc-E1, vAc-M1 andvAc-N1, respectively, and incubated for 72 hr. Total protein sampleswere run on SDS-PAGE and analyzed by western-blotting using piganti-PRRSV serum. The recombinant protein E expressed in insect cellswas detected as multi-band species of 16,18, 20, 24, and 26 kDa. The Eexpressed in insect cells showed more diversity and lower M_(r) comparedwith the native E, 26 kDa species, in the purified PRRSV. The Mexpressed in insect cells was detected as a 19 kDa band, whichcorresponded to the native M in purified PRRSV. The N expressed ininsect cells was detected as a 15 kDa band, which also corresponded tothe native N in the purified PRRSV. These specific bands were notdetected in normal insect cells (results not shown) and those infectedwith wt AcMNPV. Purified PRRS virions were analyzed in the same gel.There were at least five bands: 15, 19, 24, 26-30 and 45 kDa. Thespecific bands detected in purified PRRS virions were not observed innormal mammalian cell controls.

[0324] Immunoprecipitation was also carried out to confirm theexpression of E, M and N in insect cells. Pig anti-PRRSV serum was usedto react with the recombinant proteins expressed in insect cells andprotein A beads were used to precipitate the antigen-antibody complex. A19 kDa band was detected in the vAc-M1 infected cells. The M_(r) ofprotein detected in insect cells infected with vAc-N1 was a 15 kDa band.In preliminary studies, only some weak bands were observed in RIP ininsect cells infected with vAc-E1 (result not shown).

[0325] Glycosylation Analysis of Baculovirus Expressed E, M, and N.

[0326] To determine if the E, M, and N expressed in insect cellsunderwent N-glycosylation, the insect cells infected with therecombinant baculoviruses were treated with tunicamycin to inhibitN-linked glycosylation. After tunicamycin treatment, the 20-26 kDaspecies were not detected in insect cells infected with the vAc-E1,while the 16 and 18 kDa bands became more abundant. In the cellsinfected with vAc-M1 and vAc-N1, no changes in M_(r) of M and N proteinswere detected after the tunicamycin treatment.

[0327] To further investigate the glycosylation of recombinant E, M andN expressed in insect cells, proteins were treated with endoglycosidasesF and H and analyzed. Following treatment, the 15 kDa band of N remainedunchanged as compared with the 15 kDa band in the purified PRRS virus.No change in the 19 kDa band of M was detected after the enzymedigestion (result not shown). In the PNGase F treated purified PRRSV, atleast two bands, 26 and 45 kDa, disappeared and two more bands, 16 and27 kDa, appeared compared to the non-treated PRRSV. In theendoglycosidase H were treated PRRSV, less amount of the species around28 kDa was detected while other bands remained unchanged. The resultthat 15 and 19 kDa bands remained unchanged after PNGase F and Htreatment was consistent with that of the tunicamycin treatment ofinsect cells infected with vAc-M1 and vAc-N1.

[0328] Immunogenecity of the Recombinant Proteins.

[0329] The recombinant proteins E, M, and N were tested forimmunogenecity by immunization of rabbits with lysates of insect cellsinfected with vAc-E1, vAc-M1 and vAc-N1. Then ELISA was carried out totest for the presence of anti-PRRSV antibodies in the rabbit serumsamples. The average titers of E, M and N immunized rabbits were 384,320 and 2,056 respectively (Table 7).

[0330] Discussion

[0331] Recombinant baculoviruses containing the genes E, M, and N ofPRRSV were constructed to express E, M, and N in insect cells. Sf9 cellswere used for the propagation of baculovirus, and High Five™ cells wereused for protein expression as protein yield in High Five™ cells wasbelieved to be higher than that in Sf9 cells (Wickham et al., 1992 andDavis et al., 1993).

[0332] Immunofluorescence analysis showed that E, M and N were expressedin insect cells infected with recombinant viruses containing those genesand showed that E and M were transported to the cell surface in insectcells. This result indicates that E and M expressed in insect cells aremembrane-associated proteins and efficiently processed inpost-translational modification. The reason for low intensity ofcytoplasmic immunofluorescence of E and M in insect cells is unclear. Itmay be due to the epitope loss or modification after fixation of theinfected insect cells. In insect cells infected with vAc-N1, onlyintense cytoplasmic immunofluorescence was observed and no surfacefluorescence was detected. This result indicated that baculovirusexpressed N was not transported to cell surface but located in thecytosol. This characteristic is consistent with its nature as a veryhydrophilic nucleocapsid protein as predicated from sequence studies(Meng et al., 1994).

[0333] The recombinant E protein showed multi-bands in immunoblotting,the bands with Mr smaller than 26 kDa were not found in the purifiedPRRSV. The E expressed in insect cells showed more diversity and lowerM, compared with the native E, 26 kDa species, in the purified PRRSV.The multi-bands may be due to differential glycosylation in insect cellsduring post-translational modification. Tunicamycin treatment eliminatedthe 20-26 kDa bands and increased the intensity of the 16 kDa band. Thepresence of the 18 kDa band after treatment could be due to O-linkedglycosylation, phosphorylation or other post-translationalmodifications. The 20-26 bands represent those of differentialN-glycosylated species of E in insect cells. The 16 kDa band may be thenon-glycosylated leader-free core protein. Preliminary studies of PNGaseF and endoglycosidase H treatment of recombinant protein E showed thatit underwent complex glycosylation. The recombinant M and N did notundergo N-linked glycosylation as both the tunicamycin and PNGase F andendoglycosidase H treatments did not alter the mobilities of the 19 and15 kDa bands. These results indicate that the recombinant protein E of20-26 kDa is N-glycosylated, and that the recombinant M and N proteinsexpressed in insect cells are not N-glycosylated. The changes inmobility after tunicamycin treatment were consistent with the presenceof two N-linked glycosylation sites in the E polypeptide as determinedfrom sequence studies (Meng et al., 1994). However, sequence studiesindicated that there are 2 and 1 potential N-linked glycosylation sitesin the M and N polypeptides, respectively. In the baculovirus expressedM and N, there was no N-linked glycosylation detected. Compared with thenative counterparts, the recombinant proteins in insect cells were muchmore abundant as seen from the immunoblot (the loading amount of therecombinant proteins was about one percent of the PRRSV lane). However,it is difficult to measure the difference without oligoclonal ormonoclonal antibodies.

[0334] For the purified PRRSV, there are at least five bands: 15, 19,24, 26-30 and 45 kDa. This result is consistent with the previousreports that there are at least three structural proteins in the PRRSVvirion (Conzelmann et al., Virology 193:329-339 (1993); Nelson et al.,J. Clin. Microbiol. 31:3184-3189 (1994) and Mardassi et al., Arch.Virol. 140:1405-1418 (1994)). The 45 kDa band in the purified PRRSV maybe the ORF3 product as reported (Kapur et al., J. Gen. Virol.77:1271-1276 (1996)). The nature of the 24, 27-30 kDa species cannot befigured out. After treatment with PNGase F and endoglycosidase H, theband pattern changed for the PRRSV sample. In the PNGase F treatedPRRSV, the 16-kDa band may represent the non-glycosylated leader-removedcore protein of E, the 27-kDa band may indicate another structuralprotein of PRRSV besides E, M and N. However, the nature of these bandsneeds to be determined by oligoclonal or monoclonal antibodies.

[0335] The results from rabbit immunization test indicated that theantibodies generated from the immunization of rabbits with therecombinant proteins could recognize the native PRRSV viral antigens.The recombinant proteins showed the same antigenicity as their nativecounterparts in PRRSV infected mammalian cells, especially therecombinant N which induced higher antibody titers in rabbits than did Eand M. TABLE 7 Rabbit antiserum titers tested with ELISA Groups ofinsect cells infected with Number of rabbits Means of titers* vAc-E1 2384 vAc-M1 2 320 vAc-N1 2 2056 

[0336] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1 108 1 2352 DNA Porcine reproductive and respiratory syndrome virus 1cctgtcattg aaccaacttt aggcctgaat tgagatgaaa tggggtctat gcaaagcctt 60tttgacaaaa ttggccaact ttttgtggat gctttcacgg agttcttggt gtccattgtt 120gatatcatta tatttttggc cattttgttt ggcttcacca tcgcaggttg gctggtggtc 180ttttgcatca gattggtttg ctccgcgata ctccgtgcgc gccctgccat tcactctgag 240caattacaga agatcctatg aggcctttct ctctcagtgc caggtggaca ttcccacctg 300gggaactaaa catcctttgg ggatgctttg gcaccataag gtgtcaaccc tgattgatga 360aatggtgtcg cgtcgaatgt accgcatcat ggaaaaagca ggacaggctg cctggaaaca 420ggtagtgagc gaggctacgc tgtctcgcat tagtagtttg gatgtggtgg ctcattttca 480gcatcttgcc gccattgaag ccgagacctg taaatatctg gcctctcggc tgcccatgct 540acaccacctg cgcatgacag ggtcaaatgt aaccatagtg tataatagta ctttgaatca 600ggtgtttgct gttttcccaa cccctggttc ccggccaaag cttcatgatt tccagcaatg 660gctaatagct gtacattcct ctatattttc ctctgttgca gcttcttgta ctctttttgt 720tgtgctgtgg ttgcgggttc caatgctacg tactgttttt ggtttccgct ggttaggggc 780aatttttctt tcgaactcac ggtgaattac acggtgtgcc cgccttgcct cacccggcaa 840gcagccgcag aggcctacga acccggcagg tccctttggt gcaggatagg gcatgatcga 900tgtggggagg acgatcatga tgaactaggg tttgtggtgc cgtctggcct ctccagcgaa 960ggccacttga ccagtgctta cgcctggttg gcgtccctgt ccttcagcta tacggcccag 1020ttccatcccg agatattcgg gatagggaat gtgagtcgag tctatgttga catcaagcac 1080caattcattt gcgctgttca tgatgggcag aacaccacct tgccccacca tgacaacatt 1140tcagccgtgc ttcagaccta ttaccagcat caggtcgacg ggggcaattg gtttcaccta 1200gaatgggtgc gtcccttctt ttcctcttgg ttggttttaa atgtctcttg gtttctcagg 1260cgttcgcctg caagccatgt ttcagttcga gtctttcaga catcaagacc aacaccaccg 1320cagcggcagg ctttgctgtc ctccaagaca tcagttgcct taggcatcgc aactcggcct 1380ctgaggcgat tcgcaaagtc cctcagtgcc gcacggcgat agggacaccc gtgtatatca 1440ctgtcacagc caatgttacc gatgagaatt atttgcattc ctctgatctt ctcatgcttt 1500cttcttgcct tttctatgct tctgagatga gtgaaaaggg atttaaggtg gtatttggca 1560atgtgtcagg catcgtggca gtgtgcgtca acttcaccag ttacgtccaa catgtcaagg 1620aatttaccca acgttccttg gtagttgacc atgtgcggct gctccatttc atgacgcccg 1680agaccatgag gtgggcaact gttttagcct gtctttttac cattctgttg gcaatttgaa 1740tgtttaagta tgttggggaa atgcttgacc gcgggctgtt gctcgcaatt gcttttttta 1800tggtgtatcg tgccgtcttg ttttgttgcg ctcgtcagcg ccaacgggaa cagcggctca 1860aatttacagc tgatttacaa cttgacgcta tgtgagctga atggcacaga ttggctagct 1920aataaatttg actgggcagt ggagtgtttt gtcatttttc ctgtgttgac tcacattgtc 1980tcttatggtg ccctcactac tagccatttc cttgacacag tcggtctggt cactgtgtct 2040accgctgggt ttgttcacgg gcggtatgtt ctgagtagca tgtacgcggt ctgtgccctg 2100gctgcgttga tttgcttcgt cattaggctt gcgaagaatt gcatgtcctg gcgctactca 2160tgtaccagat ataccaactt tcttctggac actaagggca gactctatcg ttggcggtcg 2220cctgtcatca tagagaaaag gggcaaagtt gaggtcgaag gtcacctgat cgacctcaaa 2280agagttgtgc ttgatggttc cgcggctacc cctgtaacca gagtttcagc ggaacaatgg 2340agtcgtcctt ag 2352 2 2349 DNA Porcine reproductive and respiratorysyndrome virus 2 cctatcattg aaccaacttt gggtctagac tgaaatgcaa tggggtccatgcaaagcctt 60 tttgacaaga tcggtcaact ttttgtggat gctttcacgg agttcttggtgtccattgtt 120 gatatcatca tatttttggc cattttgttt ggcttcacca ttgccggctggctggtggtc 180 ttttgcatca gattggtttg ctccgcgata ctccgtgcgc gccctgccattcaccctgag 240 caattacaga agatcctatg aggcctttct ttctcagtgc caggtggacattcccgcctg 300 gggaacaaga catcctttag ggatgctttg gcaccacaag gtgtcaaccctgattgatga 360 aatggtgtcg cgtcgaatgt accgcatcat ggaaaaagca ggacaggctgcctggaaaca 420 ggtggtgagt gaggctacgc tgtctcgcat tagtggtttg gatgtggtggcccattttca 480 gcaccttgcc gccattgaag ccgagacttg taaatatttg gcctctcggttgcccatgct 540 acacaacctg cgtattacag ggtcaaatgt aaccatagtg cataatagtactttgaatca 600 ggtgtttgct attttcccaa cccccggttc tcggccaaag ctccatgattttcagcaatg 660 gctaatagct gtacattcct cgatatcctc ctctgttgca gcttcttgtactctttttgt 720 tgtgttgtgg ttacggatgc caatgctacg ttctgttttt ggtttccgctggttaggggc 780 aatttttcct tcgagctcat ggtgaattac acggtgtgcc caccttgcctcacccggcaa 840 gcagccgcac agatctacga acccaacagg tctctttggt gcaggatcgggaatgatcga 900 tgtggtgagg acgatcacga cgaactagga tttacagtac cgcctggcctctccaaagaa 960 gtccatttga ccagtgttta cgcctggttg gcgtttctgt ccttcagtaacacggcccag 1020 tttcatcccg agatattcgg aatagggaat gtgagtaagg tctatgttgacatcaatcat 1080 caactcattt gtgctgttca tgacgggcag aacaccacct tgcctcgccatgacaacatt 1140 tctgccgtgt ttcagaccta ttaccaacac caagtcgatg gtggcaactggtttcaccta 1200 gaatggctgc gtcccttctt ttcctcttgg ttggttttga atgtctcctggtttctcagg 1260 cgttcgcctg caagccatgt ttcagttcga gtctttcaga catcaagaccaacaccaccg 1320 cggcagcaaa tttcgctgtc ctccaggaca tcggctgcct taggcatggcaactcgacca 1380 ctgaggcgtt tcgcaaaatc cctcagtgcc gcacggcgat agggacacccgtgtatatca 1440 ctatcacagc caatgtaaca gatgagaact atttgcattc ttctgatcttctcatgcttt 1500 cctcttgcct tttctacgct tctgagatga gtgaaaaggg gtttaaggtggtgtttggca 1560 atgtgtcagg caccgtggct gtgtgcatca attttaccag ctatgtccaacacgtcaagg 1620 agtttaccca acgctcctta gtggtcgacc atgtgcggct gctccatttcatgacacctg 1680 aaactatgag gtgggcaact gttttagcct gtcttttcgc cattctgttggcaatttgaa 1740 tgtttaagta tgttggggaa atgcttgacc gcgggctgtt gctcgcgatcgctttttttg 1800 tggtgtatcg tgccgttctg tcttgctgcg ctcgtcagcg ccaacaacagcagctcccat 1860 ttacagttga tttataacct gacgctatgt gagctgaatg gcacagactggctggctaat 1920 aaatttgatt gggcagtgga gagttttgtc atctttcccg tgttgactcacattgtttcc 1980 tatggtgcac tcaccaccag ccatttcctt gacacagtcg gtctggttactgtgtctacc 2040 gccgggtttc atcacgggcg gtatgttctg agtagcatct acgcggtctgtgccctggct 2100 gcgtttattt gcttcgtcat taggtttgcg aagaactgca tgtcctggcgctactcttgt 2160 accagatata ccaacttcct tctggacact aagggcagcc tctatcgttggcggtcacct 2220 gtcatcatag agaaaggggg taaggttgag gtcgaaggtc atctgatcgacctaaaaaaa 2280 gttgtgcttg atggttccgc ggcaacccct ttaaccagag tttcagcggaacaatggggt 2340 cgtccctag 2349 3 2352 DNA Porcine reproductive andrespiratory syndrome virus 3 cctatcattg aaccaacttt aggcctgaat tgaaatgaaatggggtctat gcaaagcctt 60 tttgacaaaa ttggccaact tttcgtggat gctttcacggagttcttggt gtccattgtt 120 gatatcatta tatttttggc cattttgttt ggcttcaccatcgccggttg gctggtggtc 180 ttttgcatca gattggtttg ctccgcgata ctccgtgcgcgccctgccat tcactctgag 240 caattacaga agatcctatg aggcctttct ttctcagtgccaggtggaca ttcccacctg 300 gggaattaaa catcctttgg ggatgctttg gcaccataaggtgtcaaccc tgattgatga 360 aatggtgtcg cgtcgaatgt accgcatcat ggaaaaagcaggacaggctg cctggaaaca 420 ggtggtgagc gaggctacgc tgtctcgcat tagtagtttggatgtggtgg ctcactttca 480 gcatcttgcc gccattgaag ccgagacctg taaatatttggcctctcggc tgcccatgct 540 acacaacctg cgcatgacag ggtcaaatgt aaccatagtgtataatagta ctttgaatca 600 ggtgcttgct attttcccaa cccctggttc ccggccaaagcttcatgatt ttcagcaatg 660 gctaatagct gtacattcct ctatattttc ctctgttgcagcttcttgta ctctttttgt 720 tgtgctgtgg ttgcgggttc caatgctacg tattgcttttggtttccgct ggttaggggc 780 aatttttctt tcgaactcac agtgaactac acggtgtgtccaccttgcct cacccggcaa 840 gcagccacag aggcctacga acctggcagg tctctttggtgcaggatagg gtatgatcgc 900 tgtggggagg acgatcatga tgaactaggg tttgtggtgccgtctggcct ctccagcgaa 960 ggccacttga ccagtgttta cgcctggttg gcgttcctgtctttcagtta cacagcccag 1020 ttccatcctg agatattcgg gatagggaat gtgagtcaagtttatgttga catcaggcat 1080 caattcattt gcgccgttca cgacgggcag aacgccactttgcctcgcca tgacaatatt 1140 tcagccgtgt tccagactta ttaccaacat caagtcgacggcggcaattg gtttcaccta 1200 gaatggctgc gtcccttctt ttcctcttgg ttggttttaaatgtctcttg gtttctcagg 1260 cgttcgcctg caagccatgt ttcagttcga gtcttgcagacattaagacc aacaccaccg 1320 cagcggcagg ctttgctgtc ctccaagaca tcagttgccttaggtatcgc aactcggcct 1380 ctgaggcgtt tcgcaaaatc cctcagtgtc gtacggcgatagggacaccc atgtatatta 1440 ctgtcacagc caatgtaacc gatgagaatt atttgcattcctctgacctt ctcatgcttt 1500 cttcttgcct tttctacgct tctgagatga gtgaaaagggatttaaagtg gtatttggca 1560 atgtgtcagg catcgtggct gtgtgcgtca actttaccagctacgtccaa catgtcaagg 1620 aatttaccca acgctccttg gtagtcgacc atgtgcggctgctccatttc atgacacctg 1680 agaccatgag gtgggcaact gttttagcct gtctttttgccattctgttg gccatttgaa 1740 tgtttaagta tgttggggaa atgcttgacc gcgggctattgctcgtcatt gctttttttg 1800 tggtgtatcg tgccgtcttg gtttgttgcg ctcgccagcgccaacagcag caacagctct 1860 catttacagt tgatttataa cttgacgcta tgtgagctgaatggcacaga ttggttagct 1920 ggtgaatttg actgggcagt ggagtgtttt gtcatttttcctgtgttgac tcacattgtc 1980 tcctatggtg ccctcaccac cagccatttc cttgacacagtcggtctggt cactgtgtct 2040 accgccggct tttcccacgg gcggtatgtt ctgagtagcatctacgcggt ctgtgccctg 2100 gctgcgttga tttgcttcgt cattaggttt acgaagaattgcatgtcctg gcgctactca 2160 tgtaccagat ataccaactt tcttctggac actaagggcagactctatcg ttggcggtcg 2220 cctgtcatca tagagaaaag gggtaaagtt gaggtcgaaggtcatctgat cgacctcaag 2280 agagttgtgc ttgatggttc cgcggcaacc cctataaccaaagtttcagc ggagcaatgg 2340 ggtcgtcctt ag 2352 4 2351 DNA Porcinereproductive and respiratory syndrome virus 4 cctgtcattg aaccaactttaggcctgaat tgaaatgaaa tgggggccat gcaaagcctt 60 tttgacaaaa ttggccaactttttgtggat gctttcacgg agttcttggt gtccattgtt 120 gatatcatta tatttttggccattttgttt ggcttcacca tcgccggttg gctggtggtc 180 ttttgcatca gattggtttgctccgcgata ctccgtgcgc gccctgccat tcactctgag 240 caattacaga agatcttatgaggcctttct ttcccagtgc caagtggaca ttcccacctg 300 gggaactaaa catcctttggggatgttgtg gcaccataag gtgtcaaccc tgattgatga 360 aatggtgtcg cgtcgaatgtaccgcatcat ggaaaaagca gggcaggctg cctggaaaca 420 ggtggtgagc gaggctacgctgtctcgcat tagtagtttg gatgtggtgg ctcattttca 480 gcatcttgct gccattgaagccgagacctg taaatatttg gcctcccggc tgcccatgct 540 acacaacctg cgcatgacagggtcaaatgt aaccatagtg tataatagta ctttgaatca 600 ggtgtttgct attttcccaacccctggttc ccggccaaag cttcatgatt ttcagcaatg 660 gttaatagct gtacattcctccatattttc ctctgttgca gcttcctgta ctctttttgt 720 tgtgctgtgg ttgcgggttccaatactacg ttctgttttt ggtttccgct ggttaggggc 780 aatttttctt tcgagctcacggtgaattac acggtgtgtc caccttgcct cacccggcaa 840 gcagccgcag agatctacgaacccggtagg tctctttggt gcaggatagg gtatgaccga 900 tgtggggagg acgatcatgacgagctaggg tttatggtac cacctggctt ctccagcgaa 960 ggccacttga ctagtgtttacgcctggttg gcgtttttgt ccttcagcta cacggcccag 1020 ttccatcccg agatattcgggatagggaac gtgagtcgag tttatgttga catcaaacat 1080 caactcatct gcgccgaacatgacgggcaa aacaccacct tgcctcgtca tgacaacatt 1140 tcagccgtgt ttcagacctattaccaacat caagtcgacg gtggcaattg gtttcaccta 1200 gaatggcttc gtcccttcttttcctcatgg ttggttttaa atgtctcttg gtttctcagg 1260 cgttcgcctg caaaccatgtttcagttcga gtcttgcaga tattaagacc aacaccaccg 1320 cagcggcaag ctttgctgtcctccaagaca tcggttgcct taggcatcgc gactcggcct 1380 ctgaggcgat tcgcaaaatccctcagtgcc gtacggcgat agggacaccc gtgtatatta 1440 ccatcacagc caatgtgaacgatgagaatt atttacattc ttctgatctc ctcatgcttt 1500 cttcttgcct tttctatgcttctgagatga gtgaaaaggg gtttaaggtg gtatttggca 1560 atgtgtcagg catcgtggctgtgtgtgtca attttaccag ctatgtccaa catgtcaggg 1620 agtttaccca acgctccttggtggtcgacc atgtgcggtt gctccatttc atgacacctg 1680 agaccatgag gtgggcaactgttttagcct gtctttttgc cattctgttg gcaatttgaa 1740 tgtttaagca tgttggggaaatgcttgacc gcgggctgtt gctcgcgatt gctttctttg 1800 tggtttatcg tgccgttctgttttgctgtg ctcgccagcg ccagcaacag cagcagctcc 1860 catctacagt tgatttataacttgacgcta tgtgagctga atggcacaga ttggttagct 1920 aataaatttg attgggcagtggagagtttt gtcatctttc ccgttttgac tcacattgtc 1980 tcctatggtg ccctcactaccagccatttc cttgacacag tcgctttagt cactgtgtct 2040 accgccgggt ttgttcacgggcggtatgtc ctgagtagca tctacgcggt ctgtgccctg 2100 gctgcgttga cttgcttcatcatcaggttt gcaaagaatt gcatgtcctg gcgctactcg 2160 tgtaccagat ataccaactttctcctggac actaagggca gactctatcg ttggcggtcg 2220 cctgtcatca tagagaaaaggggcaaagtt gaggtcgaag gtcactgatc gacctcaaaa 2280 gagttgtgct tgatggttccgtggcaaccc ctataaccag agattcagcg gaacaatggg 2340 gtcgtcctta g 2351 52352 DNA Porcine reproductive and respiratory syndrome virus 5cctgtcattg aaccaacttt aggcctgaat tgaaatgaaa tggggtccat gcaaagcctt 60tttgacaaaa ttggccaact ttttgtggat gctttcacgg agttcttggt gtccattgtt 120gatatcatta tattcttggc cattttgttt ggcttcacca tcgccggttg gctggtggtc 180ttttgcatca gattggtttg ctccgcgata ctccgtacgc gccctgccat tcactctgag 240caattacaga agatcttatg aggcctttct ttcccagtgc caagtggaca ttcccacctg 300gggaactaaa catcctttgg ggatgttttg gcaccataag gtgtcaaccc tgattgatga 360gatggtgtcg cgtcgaatgt accgcatcat ggaaaaagca ggacaggctg cctggaaaca 420ggtggtgagc gaggctacgc tgtctcgcat tagtagtttg gatgtggtgg ctcattttca 480gcatcttgcc gccatcgaag ccgagacctg taaatatttg gcctcccggc tgcccatgct 540acacaacctg cgcatgacag ggtcaaatgt aaccatagtg tataatagta ctttgaatcg 600ggtgtttgct attttcccaa cccctggttc ccggccaaag cttcatgact ttcagcaatg 660gctaatagct gtgcattcct ccatattttc ctctgttgca gcttcttgta ctctctttgt 720tgtgctgtgg ttgcgggttc caatactacg tactgttttt ggtttccgct ggttaggggc 780aatttttctt tcgaactcat agtgaattac acggtgtgcc caccttgcct cacccggcaa 840gcagccgcag aggcctacga acccggtagg tctctttggt gcaggatagg gtacgatcga 900tgtggagagg acgaccatga cgagctaggg tttatgatac cgtctggcct ctccagcgaa 960ggccacttga ccagtgttta cgcctggttg gcgttcttgt ccttcagcta cacggcccag 1020ttccaccccg agatattcgg gatagggaat gtgagtcgag tttatgttga catcaaacat 1080caactcatct gcgccgaaca tgacgggcag aacaccacct tgcctcgtca tgacaacatt 1140tcggccgtgt ttcagaccta ttaccaacat caagtcgacg gcggcaattg gtttcaccta 1200gaatggctgc gtcccttctt ttcctcatgg ttggttttaa atgtctcttg gtttctcagg 1260cgttcgcctg caaaccatgt ttcagttcga gtcttgcaga cattaagacc aacaccaccg 1320cagcggcaag ctttgctgtc ctccaagaca tcagttgcct taggcatcgc aactcggcct 1380ctgaggcgat tcgcaaaatc cctcagtgcc gtacggcgat agggacacct atgtatatta 1440ccatcacagc caatgtgaca gatgaaaatt atttacattc ttctgatctc ctcatgctct 1500cttcttgcct tttctatgct tctgagatga gtgaaaaggg atttgaggtg gtttttggca 1560atgtgtcagg catcgtggct gtgtgtgtca attttaccag ctacgttcaa catgtcaggg 1620agtttaccca acgctccttg atggtcgacc atgtgcggct gctccatttc atgacacctg 1680agaccatgag gtgggcaacc gttttagcct gtctttttgc tattctgttg gcaatttgaa 1740tgtttaagta tgttggggaa atgcttgacc gtgggctgtt gctcgcgatt gctttctttg 1800tggtgtatcg tgccgttctg ttttactgtg ctcgccgacg cccacagcaa cagcagctct 1860catctgcaat tgatttacaa cttgacgcta tgtgagctga atggcacaga ttggctagct 1920gatagatttg attgggcagt ggagagcttt gtcatctttc ctgttttgac tcacattgtc 1980tcctatggcg ccctcaccac cagccatttc cttgacacaa ttgctttagt cactgtgtct 2040accgccgggt ttgttcacgg gcggtatgtc ctaagtagca tctacgcggt ctgtgccctg 2100gctgcgttga cttgcttcgt cattaggttt gtgaagaatt gcatgtcctg gcgctactca 2160tgtactagat ataccaactt tcttctggat actaagggca gactctatcg ttggcggtcg 2220cctgtcatca tagagaagag gggcaaagtt gaggtcgaag gtcatctgat cgatctcaaa 2280agagttgtgc ttgatggttc cgtggcaacc cctataacca gagtttcagc ggaacaatgg 2340ggtcgtcctt ag 2352 6 2352 DNA Porcine reproductive and respiratorysyndrome virus 6 cctgtcattg aaccaacttt aggcctgaat tgagatgaaa tggggtctatgcaaagcctt 60 tttgacaaaa ttggccaact ttttgtggat gctttcacgg agttcttggtgtccattgtt 120 gatatcatta tatttttggc cattttgttt ggcttcacca tcgcaggttggctggtggtc 180 ttttgcatca gattggtttg ctccgcgata ctccgtgcgc gccctgccattcactctgag 240 caattacaga agatcctatg aggcctttct ctctcagtgc caggtggacattcccacctg 300 gggaactaaa catcctttgg ggatgctttg gcaccataag gtgtcaaccctgattgatga 360 aatggtgtcg cgtcgaatgt accgcatcat ggaaaaagca ggacaggctgcctggaaaca 420 ggtagtgagc gaggctacgc tgtctcgcat tagtagtttg gatgtggtggctcattttca 480 gcatcttgcc gccattgaag ccgagacctg taaatatctg gcctctcggctgcccatgct 540 acaccacctg cgcatgacag ggtcaaatgt aaccatagtg tataatagtactttgaatca 600 ggtgtttgct gttttcccaa cccctggttc ccggccaaag cttcatgatttccagcaatg 660 gctaatagct gtacattcct ctatattttc ctctgttgca gcttcttgtactctttttgt 720 tgtgctgtgg ttgcgggttc caatgctacg tactgttttt ggtttccgctggttaggggc 780 aatttttctt tcgaactcac ggtgaattac acggtgtgcc cgccttgcctcacccggcaa 840 gcagccgcag aggcctacga acccggcagg tccctttggt gcaggatagggcatgatcga 900 tgtggggagg acgatcatga tgaactaggg tttgtggtgc cgtctggcctctccagcgaa 960 ggccacttga ccagtgctta cgcctggttg gcgtccctgt ccttcagctatacggcccag 1020 ttccatcccg agatattcgg gatagggaat gtgagtcgag tctatgttgacatcaagcac 1080 caattcattt gcgctgttca tgatgggcag aacaccacct tgccccaccatgacaacatt 1140 tcagccgtgc ttcagaccta ttaccagcat caggtcgacg ggggcaattggtttcaccta 1200 gaatgggtgc gtcccttctt ttcctcttgg ttggttttaa atgtctcttggtttctcagg 1260 cgttcgcctg caagccatgt ttcagttcga gtctttcaga catcaagaccaacaccaccg 1320 cagcggcagg ctttgctgtc ctccaagaca tcagttgcct taggcatcgcaactcggcct 1380 ctgaggcgat tcgcaaagtc cctcagtgcc gcacggcgat agggacacccgtgtatatca 1440 ctgtcacagc caatgttacc gatgagaatt atttgcattc ctctgatcttctcatgcttt 1500 cttcttgcct tttctatgct tctgagatga gtgaaaaggg atttaaggtggtatttggca 1560 atgtgtcagg catcgtggca gtgtgcgtca acttcaccag ttacgtccaacatgtcaagg 1620 aatttaccca acgttccttg gtagttgacc atgtgcggct gctccatttcatgacgcccg 1680 agaccatgag gtgggcaact gttttagcct gtctttttac cattctgttggcaatttgaa 1740 tgtttaagta tgttggggaa atgcttgacc gcgggctgtt gctcgcaattgcttttttta 1800 tggtgtatcg tgccgtcttg ttttgttgcg ctcgtcagcg ccaacgggaacagcggctca 1860 aatttacagc tgatttacaa cttgacgcta tgtgagctga atggcacagattggctagct 1920 aataaatttg actgggcagt ggagtgtttt gtcatttttc ctgtgttgactcacattgtc 1980 tcttatggtg ccctcactac tagccatttc cttgacacag tcggtctggtcactgtgtct 2040 accgctgggt ttgttcacgg gcggtatgtt ctgagtagca tgtacgcggtctgtgccctg 2100 gctgcgttga tttgcttcgt cattaggctt gcgaagaatt gcatgtcctggcgctactca 2160 tgtaccagat ataccaactt tcttctggac actaagggca gactctatcgttggcggtcg 2220 cctgtcatca tagagaaaag gggcaaagtt gaggtcgaag gtcacctgatcgacctcaaa 2280 agagttgtgc ttgatggttc cgcggctacc cctgtaacca gagtttcagcggaacaatgg 2340 agtcgtcctt ag 2352 7 2352 DNA Porcine reproductive andrespiratory syndrome virus 7 cccgtcattg aaccaacttt aggcctgaat tgaaatgaaatggggtccgt gcaaagcctt 60 tttgacaaaa ttggccaact ttttgtggat gctttcacggagttcctggt gtccattgtt 120 gatatcatca tatttttggc cattttgttt ggcttcaccatcgccggttg gctggtggtc 180 ttttgcatca gattggtttg ctccgcgata ctccgtacgcgccctgccat tcactctgag 240 caattacaga agatcttatg aggccttttt atcccagtgccaagtggaca ttcccacctg 300 gggaactaaa catcctttgg ggatgttttg gcaccataaggtgtcaaccc tgattgatga 360 aatggtgtcg cgtcgcatgt accgcatcat ggaaaaagcagggcaggctg cctggaaaca 420 ggtggtgagc gaggctacgc tgtcccgcat tagtagtttggatgtggtgg ctcattttca 480 gcatcttgcc gccattgaag ccgagacttg taaatatttggcctcccggc tgcccatgct 540 acataacctg cgcataacag ggtcaaatgt aaccatagtgtataatagta cttcggagca 600 ggtgtttgct attttcccaa cccctggttc ccggccaaagcttcatgatt ttcagcaatg 660 gttaatagct gtacattcct ccatattttc ctctgttgcagcttcttgta ctctttttgt 720 tgtgctgtgg ctgcgggttc caatgctacg tactgtttttggtttccgct ggttaggggg 780 aatttttcct tcgaactcat ggtgaattac acggtgtgtccaccttgcct cacccggcaa 840 gcagccgcag aggtctacga acccggtagg tctctttggtgcaggatagg gtatgaccga 900 tgtggggagg acgatcatga cgagctaggg tttatgataccgcctggcct ctccagcgaa 960 ggccacttga ctagtgttta cgcctggttg gcgtttttgtccttcagcta cacggcccag 1020 ttccatcccg agatattcgg gatagggaat gtgagtcgagtttatgttga catcaaacat 1080 caactcattt gcgccgaaca tgacggacag aacgccaccttgcctcgtca tgacaatatt 1140 tcagccgtgt ttcagaccta ttaccaacat caagtcgatggcggcaattg gtttcaccta 1200 gaatggcttc gtcccttctt ttcctcatgg ttggttttaaatgtctcttg gtatctcagg 1260 cgttcgcctg caaaccatgc ttcagttcga gtcttgcagatattaagacc aacactaccg 1320 cagcggcaag ctttgctgtc ctccaagaca tcagttgccttaggcatcgc aactcggcct 1380 ctgaggcgat tcgcaaaatc cctcagtgcc gtacggcgatagggacaccc gtgtatatta 1440 ccatcacagc caatgtgaca gatgagaatt atttacattcttctgatctc ctcatgcttt 1500 cttcttgcct tttctacgct tctgagatga gtgaaaaaggattcaaggtg gtatttggca 1560 atgtgtcagg catcgtggct gtgtgtgtca attttaccagctacgtccaa catgtcaggg 1620 agtttaccca acgctccctg gtggtcgacc atgtgcggttgctccatttc atgacacctg 1680 aaaccatgag gtgggcaact gttttagcct gtctttttgccattctgctg gcaatttgaa 1740 tgtttaagta tgttggggaa atgcttgacc gcgggctgttgctcgcgatt gctttctttg 1800 tggtgtatcg tgccgttctg ttttgctgtg ctcgccaacgccagcgccaa cagcagctcc 1860 catctacagc tgatttacaa cttgacgcta tgtgagctgaatggcacaga ttggctagct 1920 gataaatttg attgggcagt ggagagtttt gtcatctttcccgttttgac tcacattgtc 1980 tcctatggtg ccctcactac tagccatctc cttgacacagtcgccttagt cactgtgtct 2040 accgccgggt ttgttcacgg gcggtatgtc ctaagtagcatctacgcggt ctgtgccctg 2100 gctgcgttag cttgcttcgt cattaggttt gcaaagaattgcatgtcctg gcgctattcg 2160 tgtaccagat ataccaactt tcttctggac actaagggcagactctatcg ttggcattcg 2220 cctgtcatca tagagaaaag gggcaaagtt gaggtcgaaggtcatctgat cgacctcaaa 2280 agagttgtgc ttgacggttc cgtggcaacc cctataaccagagtttcagc ggaacaatgg 2340 ggtcgtcctt ag 2352 8 256 PRT Porcinereproductive and respiratory syndrome virus 8 Met Lys Trp Gly Leu CysLys Ala Phe Leu Thr Lys Leu Ala Asn Phe 1 5 10 15 Leu Trp Met Leu SerArg Ser Ser Trp Cys Pro Leu Leu Ile Ser Leu 20 25 30 Tyr Phe Trp Pro PheCys Leu Ala Ser Pro Ser Gln Val Gly Trp Trp 35 40 45 Ser Phe Ala Ser AspTrp Phe Ala Pro Arg Tyr Ser Val Arg Ala Leu 50 55 60 Pro Phe Thr Leu SerAsn Tyr Arg Arg Ser Tyr Glu Ala Phe Leu Ser 65 70 75 80 Gln Cys Gln ValAsp Ile Pro Thr Trp Gly Thr Lys His Pro Leu Gly 85 90 95 Met Leu Trp HisHis Lys Val Ser Thr Leu Ile Asp Glu Met Val Ser 100 105 110 Arg Arg MetTyr Arg Ile Met Glu Lys Ala Gly Gln Ala Ala Trp Lys 115 120 125 Gln ValVal Ser Glu Ala Thr Leu Ser Arg Ile Ser Ser Leu Asp Val 130 135 140 ValAla His Phe Gln His Leu Ala Ala Ile Glu Ala Glu Thr Cys Lys 145 150 155160 Tyr Leu Ala Ser Arg Leu Pro Met Leu His Met Leu Arg Met Thr Gly 165170 175 Ser Asn Val Thr Ile Val Tyr Asn Ser Thr Leu Asn Gln Val Phe Ala180 185 190 Val Phe Pro Thr Pro Gly Ser Arg Pro Lys Leu His Asp Phe GlnGln 195 200 205 Trp Leu Ile Ala Val His Ser Ser Ile Phe Ser Ser Val AlaAla Ser 210 215 220 Cys Thr Leu Phe Val Val Leu Trp Leu Arg Val Pro MetLeu Arg Thr 225 230 235 240 Val Phe Gly Phe Arg Trp Leu Gly Ala Ile PheLeu Ser Asn Ser Arg 245 250 255 9 256 PRT Porcine reproductive andrespiratory syndrome virus 9 Met Lys Trp Gly Pro Cys Lys Ala Phe Leu ThrLys Leu Ala Asn Phe 1 5 10 15 Leu Trp Met Leu Ser Arg Ser Ser Trp CysPro Leu Leu Ile Ser Leu 20 25 30 Tyr Phe Trp Pro Phe Cys Leu Ala Ser ProSer Pro Val Gly Trp Trp 35 40 45 Ser Phe Ala Ser Asp Trp Phe Ala Pro ArgTyr Ser Val Arg Ala Leu 50 55 60 Pro Phe Thr Leu Ser Asn Tyr Arg Arg SerTyr Glu Ala Phe Leu Ser 65 70 75 80 Gln Cys Gln Val Asp Ile Pro Thr TrpGly Thr Lys His Pro Leu Gly 85 90 95 Met Leu Trp His His Lys Val Ser ThrLeu Ile Asp Glu Met Val Ser 100 105 110 Arg Arg Met Tyr Arg Ile Met GluLys Ala Gly Gln Ala Ala Trp Lys 115 120 125 Gln Val Val Ser Glu Ala ThrLeu Ser Arg Ile Ser Ser Leu Asp Val 130 135 140 Val Ala His Phe Gln HisLeu Ala Ala Ile Glu Ala Glu Thr Cys Lys 145 150 155 160 Tyr Leu Ala SerArg Leu Pro Met Leu His Asn Leu Arg Met Thr Gly 165 170 175 Ser Asn ValThr Ile Val Tyr Asn Ser Thr Leu Asn Gln Val Phe Ala 180 185 190 Ile PhePro Thr Pro Gly Ser Arg Pro Lys Leu His Asp Phe Gln Gln 195 200 205 TrpLeu Ile Ala Val His Ser Ser Ile Phe Ser Ser Val Ala Ala Ser 210 215 220Cys Thr Leu Phe Val Val Leu Trp Leu Arg Val Pro Ile Leu Arg Ser 225 230235 240 Val Phe Gly Phe Arg Trp Leu Gly Ala Ile Phe Leu Ser Ser Ser Arg245 250 255 10 255 PRT Porcine reproductive and respiratory syndromevirus 10 Met Lys Trp Gly Pro Cys Lys Ala Phe Leu Thr Lys Leu Ala Asn Phe1 5 10 15 Leu Trp Met Leu Ser Arg Ser Ser Trp Cys Pro Leu Leu Ile SerLeu 20 25 30 Ser Phe Trp Pro Phe Cys Leu Ala Ser Pro Ser Pro Val Gly TrpTrp 35 40 45 Ser Phe Ala Ser Asp Trp Phe Ala Pro Arg Tyr Ser Val Arg AlaLeu 50 55 60 Pro Phe Thr Leu Ser Asn Tyr Arg Arg Ser Tyr Glu Ala Phe LeuSer 65 70 75 80 Gln Cys Gln Val Asp Ile Pro Thr Trp Gly Thr Lys His ProLeu Gly 85 90 95 Met Phe Trp His His Lys Val Ser Thr Leu Ile Asp Glu MetVal Ser 100 105 110 Arg Arg Met Tyr Arg Ile Met Glu Lys Ala Gly Gln AlaAla Trp Lys 115 120 125 Gln Val Val Ser Glu Ala Thr Leu Ser Arg Ile SerSer Leu Asp Val 130 135 140 Val Ala His Phe Gln His Leu Ala Ala Ile GluAla Glu Thr Cys Lys 145 150 155 160 Tyr Leu Ala Ser Arg Leu Pro Met LeuHis Asn Leu Arg Met Thr Gly 165 170 175 Ser Asn Val Thr Ile Val Tyr AsnSer Thr Leu Asn Arg Val Phe Ala 180 185 190 Ile Phe Pro Thr Pro Gly SerArg Pro Lys Leu His Asp Phe Gln Gln 195 200 205 Trp Leu Ile Ala Val HisSer Ser Ile Phe Ser Ser Val Ala Ala Ser 210 215 220 Cys Thr Leu Phe ValVal Leu Trp Leu Arg Val Pro Ile Leu Arg Thr 225 230 235 240 Val Phe GlyPhe Arg Trp Leu Gly Ala Ile Phe Leu Ser Asn Ser 245 250 255 11 256 PRTPorcine reproductive and respiratory syndrome virus 11 Met Lys Trp GlyLeu Cys Lys Ala Phe Leu Thr Lys Leu Ala Asn Phe 1 5 10 15 Ser Trp MetLeu Ser Arg Ser Ser Trp Cys Pro Leu Leu Ile Ser Leu 20 25 30 Tyr Phe TrpPro Phe Cys Leu Ala Ser Pro Ser Pro Val Gly Trp Trp 35 40 45 Ser Phe AlaSer Asp Trp Phe Ala Pro Arg Tyr Ser Val Arg Ala Leu 50 55 60 Pro Phe ThrLeu Ser Asn Tyr Arg Arg Ser Tyr Glu Ala Phe Leu Ser 65 70 75 80 Gln CysGln Val Asp Ile Pro Thr Trp Gly Ile Lys His Pro Leu Gly 85 90 95 Met PheTrp His His Lys Val Ser Thr Leu Ile Asp Glu Met Val Ser 100 105 110 ArgArg Met Tyr Arg Ile Met Glu Lys Ala Gly Gln Ala Ala Trp Lys 115 120 125Gln Val Val Ser Glu Ala Thr Leu Ser Arg Ile Ser Ser Leu Asp Val 130 135140 Val Ala His Phe Gln His Leu Ala Ala Ile Glu Ala Glu Thr Cys Lys 145150 155 160 Tyr Leu Ala Ser Arg Leu Pro Met Leu His Asn Leu Arg Met ThrGly 165 170 175 Ser Asn Val Thr Ile Val Tyr Asn Ser Thr Leu Asn Gln ValLeu Ala 180 185 190 Ile Phe Pro Thr Pro Gly Ser Arg Pro Lys Leu His AspPhe Gln Gln 195 200 205 Trp Leu Ile Ala Val His Ser Ser Ile Phe Ser SerVal Ala Ala Ser 210 215 220 Cys Thr Leu Phe Val Val Leu Trp Leu Arg ValPro Met Leu Arg Ile 225 230 235 240 Ala Phe Gly Phe Arg Trp Leu Gly AlaIle Phe Leu Ser Asn Ser Gln 245 250 255 12 256 PRT Porcine reproductiveand respiratory syndrome virus 12 Met Lys Trp Gly Pro Cys Lys Ala PheLeu Thr Lys Leu Ala Asn Phe 1 5 10 15 Leu Trp Met Leu Ser Arg Ser SerTrp Cys Pro Leu Leu Ile Ser Ser 20 25 30 Tyr Phe Trp Pro Phe Cys Leu AlaSer Pro Ser Pro Val Gly Trp Trp 35 40 45 Ser Phe Ala Ser Asp Trp Phe AlaPro Arg Tyr Ser Val Arg Ala Leu 50 55 60 Pro Phe Thr Leu Ser Asn Tyr ArgArg Ser Tyr Glu Ala Phe Leu Ser 65 70 75 80 Gln Cys Gln Val Asp Ile ProThr Trp Gly Thr Lys His Pro Leu Gly 85 90 95 Met Phe Trp His His Lys ValSer Thr Leu Ile Asp Glu Met Val Ser 100 105 110 Arg Arg Met Tyr Arg IleMet Glu Lys Ala Gly Gln Ala Ala Trp Lys 115 120 125 Gln Val Val Ser GluAla Thr Leu Ser Arg Ile Ser Ser Leu Asp Val 130 135 140 Val Ala His PheGln His Leu Ala Ala Ile Glu Ala Glu Thr Cys Lys 145 150 155 160 Tyr LeuAla Ser Arg Leu Pro Met Leu His Asn Leu Arg Ile Thr Gly 165 170 175 SerAsn Val Thr Ile Val Tyr Asn Ser Thr Ser Glu Gln Val Phe Ala 180 185 190Ile Phe Pro Thr Pro Gly Ser Arg Pro Lys Leu His Asp Phe Gln Gln 195 200205 Trp Leu Ile Ala Val His Ser Ser Ile Phe Ser Ser Val Ala Ala Ser 210215 220 Cys Thr Leu Phe Val Val Leu Trp Leu Arg Val Pro Met Leu Arg Thr225 230 235 240 Val Phe Gly Phe Arg Trp Leu Gly Gly Ile Phe Pro Ser AsnSer Trp 245 250 255 13 256 PRT Porcine reproductive and respiratorysyndrome virus 13 Met Gln Trp Gly Pro Cys Lys Ala Phe Leu Thr Arg SerVal Asn Phe 1 5 10 15 Leu Trp Met Leu Ser Arg Ser Ser Trp Cys Pro LeuLeu Ile Ser Ser 20 25 30 Tyr Phe Trp Pro Phe Cys Leu Ala Ser Pro Leu ProAla Gly Trp Trp 35 40 45 Ser Phe Ala Ser Asp Trp Phe Ala Pro Arg Tyr SerVal Arg Ala Leu 50 55 60 Pro Phe Thr Leu Ser Asn Tyr Arg Arg Ser Tyr GluAla Phe Leu Ser 65 70 75 80 Gln Cys Gln Val Asp Ile Pro Ala Trp Gly ThrArg His Pro Leu Gly 85 90 95 Met Leu Trp His His Lys Val Ser Thr Leu IleAsp Glu Met Val Ser 100 105 110 Arg Arg Met Tyr Arg Ile Met Glu Lys AlaGly Gln Ala Ala Trp Lys 115 120 125 Gln Val Val Ser Glu Ala Thr Leu SerArg Ile Ser Gly Leu Asp Val 130 135 140 Val Ala His Phe Gln His Leu AlaAla Ile Glu Ala Glu Thr Cys Lys 145 150 155 160 Tyr Leu Ala Ser Arg LeuPro Met Leu His Asn Leu Arg Ile Thr Gly 165 170 175 Ser Asn Val Thr IleVal His Asn Ser Thr Leu Asn Gln Val Phe Ala 180 185 190 Ile Phe Pro ThrPro Gly Ser Arg Pro Lys Leu His Asp Phe Gln Gln 195 200 205 Trp Leu IleAla Val His Ser Ser Ile Ser Ser Ser Val Ala Ala Ser 210 215 220 Cys ThrLeu Phe Val Val Leu Trp Leu Arg Met Pro Met Leu Arg Ser 225 230 235 240Val Phe Gly Phe Arg Trp Leu Gly Ala Ile Phe Pro Ser Ser Ser Trp 245 250255 14 256 PRT Porcine reproductive and respiratory syndrome virus 14Met Lys Trp Gly Pro Cys Lys Ala Phe Leu Thr Lys Leu Ala Asn Phe 1 5 1015 Leu Trp Met Leu Ser Arg Ser Ser Trp Cys Pro Leu Leu Ile Ser Leu 20 2530 Tyr Phe Trp Pro Phe Cys Leu Ala Ser Pro Ser Pro Val Gly Trp Trp 35 4045 Ser Phe Ala Ser Asp Trp Phe Ala Pro Arg Tyr Ser Val Arg Ala Leu 50 5560 Pro Phe Thr Leu Ser Asn Tyr Arg Arg Ser Tyr Glu Ala Phe Leu Ser 65 7075 80 Gln Cys Gln Val Asp Ile Pro Thr Trp Gly Thr Lys His Pro Leu Gly 8590 95 Met Leu Trp His His Lys Val Ser Thr Leu Ile Asp Glu Met Val Ser100 105 110 Arg Arg Met Tyr Arg Ile Met Glu Lys Ala Gly Gln Ala Ala TrpLys 115 120 125 Gln Val Val Ser Glu Ala Thr Leu Ser Arg Ile Ser Ser LeuAsp Val 130 135 140 Val Ala His Phe Gln His Leu Ala Ala Ile Glu Ala GluThr Cys Lys 145 150 155 160 Tyr Leu Ala Ser Arg Leu Pro Met Leu His AsnLeu Arg Met Thr Gly 165 170 175 Ser Asn Val Thr Ile Val Tyr Asn Ser ThrLeu Asn Gln Val Phe Ala 180 185 190 Ile Phe Pro Thr Pro Gly Ser Arg ProLys Leu His Asp Phe Gln Gln 195 200 205 Trp Leu Ile Ala Val His Ser SerIle Phe Ser Ser Val Ala Ala Ser 210 215 220 Cys Thr Leu Phe Val Val LeuTrp Leu Arg Val Pro Ile Leu Arg Thr 225 230 235 240 Val Phe Gly Phe ArgTrp Leu Gly Ala Ile Phe Leu Ser Asn Ser Gln 245 250 255 15 249 PRTPorcine reproductive and respiratory syndrome virus 15 Met Gln Trp GlyHis Cys Gly Val Lys Ser Ala Ser Cys Ser Trp Thr 1 5 10 15 Pro Ser LeuSer Ser Leu Leu Val Trp Leu Ile Leu Pro Phe Ser Leu 20 25 30 Pro Tyr CysLeu Gly Ser Pro Ser Gln Asp Gly Tyr Trp Ser Phe Phe 35 40 45 Ser Glu TrpPhe Ala Pro Arg Phe Ser Val Arg Ala Leu Pro Phe Thr 50 55 60 Leu Pro AsnTyr Arg Arg Ser Tyr Glu Gly Leu Leu Pro Asn Cys Arg 65 70 75 80 Pro AspVal Pro Gln Phe Ala Val Lys His Pro Leu Gly Met Phe Trp 85 90 95 His MetArg Val Ser His Leu Ile Asp Glu Met Val Ser Arg Arg Ile 100 105 110 TyrGln Thr Met Glu His Ser Gly Gln Ala Ala Trp Lys Gln Val Val 115 120 125Gly Glu Ala Thr Leu Thr Lys Leu Ser Gly Leu Asp Ile Val Thr His 130 135140 Phe Gln His Leu Ala Ala Val Glu Ala Asp Ser Cys Arg Phe Leu Ser 145150 155 160 Ser Arg Leu Val Met Leu Lys Asn Leu Ala Val Gly Asn Val SerLeu 165 170 175 Gln Tyr Asn Thr Thr Leu Asp Arg Val Glu Leu Ile Phe ProThr Pro 180 185 190 Gly Thr Arg Pro Lys Leu Thr Asp Phe Arg Gln Trp LeuIle Ser Val 195 200 205 His Ala Ser Ile Phe Ser Ser Val Ala Ser Ser ValThr Leu Phe Ile 210 215 220 Val Leu Trp Leu Arg Ile Pro Ala Leu Arg TyrVal Phe Gly Phe His 225 230 235 240 Trp Pro Thr Ala Thr His His Ser Ser245 16 254 PRT Porcine reproductive and respiratory syndrome virus 16Met Ala Asn Ser Cys Thr Phe Leu Tyr Ile Phe Leu Cys Cys Ser Phe 1 5 1015 Leu Tyr Ser Phe Cys Cys Ala Val Val Ala Gly Ser Asn Ala Thr Tyr 20 2530 Cys Phe Trp Phe Pro Leu Val Arg Gly Asn Phe Ser Phe Glu Leu Thr 35 4045 Val Asn Tyr Thr Val Cys Pro Pro Cys Leu Thr Arg Gln Ala Ala Ala 50 5560 Glu Ala Tyr Glu Pro Gly Arg Ser Leu Trp Cys Arg Ile Gly His Asp 65 7075 80 Arg Cys Gly Glu Asp Asp His Asp Glu Leu Gly Phe Val Val Pro Ser 8590 95 Gly Leu Ser Ser Glu Gly His Leu Thr Ser Ala Tyr Ala Trp Leu Ala100 105 110 Ser Leu Ser Phe Ser Tyr Thr Thr Gln Phe His Pro Glu Ile PheGly 115 120 125 Ile Gly Asn Val Ser Arg Val Tyr Val Asp Ile Lys His GlnPhe Ile 130 135 140 Cys Ala Val His Asp Gly Gln Asn Thr Thr Leu Pro HisHis Asp Asn 145 150 155 160 Ile Ser Ala Val Leu Gln Thr Tyr Tyr Gln HisGln Val Asp Gly Gly 165 170 175 Asn Trp Phe His Leu Glu Trp Val Arg ProPhe Phe Ser Ser Trp Leu 180 185 190 Val Leu Asn Val Ser Trp Phe Leu ArgArg Ser Pro Ala Ser His Val 195 200 205 Ser Val Arg Val Phe Gln Thr SerArg Pro Thr Pro Pro Gln Arg Gln 210 215 220 Ala Leu Leu Ser Ser Lys ThrSer Val Ala Leu Gly Ile Ala Thr Arg 225 230 235 240 Pro Leu Arg Arg PheAla Lys Ser Leu Ser Ala Ala Arg Arg 245 250 17 254 PRT Porcinereproductive and respiratory syndrome virus 17 Met Ala Asn Ser Cys ThrPhe Leu Tyr Ile Phe Leu Cys Cys Ser Phe 1 5 10 15 Leu Tyr Ser Phe CysCys Ala Val Val Ala Gly Ser Asn Ala Thr Tyr 20 25 30 Cys Phe Trp Phe ProLeu Val Arg Gly Asn Phe Ser Phe Glu Leu Thr 35 40 45 Val Asn Tyr Thr ValCys Pro Pro Cys Leu Thr Arg Gln Ala Ala Thr 50 55 60 Glu Ala Tyr Glu ProGly Arg Ser Leu Trp Cys Arg Ile Gly Tyr Asp 65 70 75 80 Arg Cys Gly GluAsp Asp His Asp Glu Leu Gly Phe Val Val Pro Ser 85 90 95 Gly Leu Ser SerGlu Gly His Leu Thr Ser Val Tyr Ala Trp Leu Ala 100 105 110 Phe Leu SerPhe Ser Tyr Thr Ala Gln Phe His Pro Glu Ile Phe Gly 115 120 125 Ile GlyAsn Val Ser Gln Val Tyr Val Asp Ile Arg His Gln Phe Ile 130 135 140 CysAla Val His Asp Gly Gln Asn Ala Thr Leu Pro Arg His Asp Asn 145 150 155160 Ile Ser Ala Val Phe Gln Thr Tyr Tyr Gln His Gln Val Asp Gly Gly 165170 175 Asn Trp Phe His Leu Glu Trp Leu Arg Pro Phe Phe Ser Ser Trp Leu180 185 190 Val Leu Asn Val Ser Trp Phe Leu Arg Arg Ser Pro Ala Ser HisVal 195 200 205 Ser Val Arg Val Leu Gln Thr Leu Arg Pro Thr Pro Pro GlnArg Gln 210 215 220 Ala Leu Leu Ser Ser Lys Thr Ser Val Ala Leu Gly IleAla Thr Arg 225 230 235 240 Pro Leu Arg Arg Phe Ala Lys Ser Leu Ser ValVal Arg Arg 245 250 18 254 PRT Porcine reproductive and respiratorysyndrome virus 18 Met Ala Asn Ser Cys Ala Phe Leu His Ile Phe Leu CysCys Ser Phe 1 5 10 15 Leu Tyr Ser Leu Cys Cys Ala Val Val Ala Gly SerAsn Thr Thr Tyr 20 25 30 Cys Phe Trp Phe Pro Leu Val Arg Gly Asn Phe SerPhe Glu Leu Ile 35 40 45 Val Asn Tyr Thr Val Cys Pro Pro Cys Leu Thr ArgGln Ala Ala Ala 50 55 60 Glu Ala Tyr Glu Pro Gly Arg Ser Leu Trp Cys ArgIle Gly Tyr Asp 65 70 75 80 Arg Cys Gly Glu Asp Asp His Asp Glu Leu GlyPhe Met Ile Pro Ser 85 90 95 Gly Leu Ser Ser Glu Gly His Leu Thr Ser ValTyr Ala Trp Leu Ala 100 105 110 Phe Leu Ser Phe Ser Tyr Thr Ala Gln PheHis Pro Glu Ile Phe Gly 115 120 125 Ile Gly Asn Val Ser Arg Val Tyr ValAsp Ile Lys His Gln Leu Ile 130 135 140 Cys Ala Glu His Asp Gly Gln AsnThr Thr Leu Pro Arg His Asp Asn 145 150 155 160 Ile Ser Ala Val Phe GlnThr Tyr Tyr Gln His Gln Val Asp Gly Gly 165 170 175 Asn Trp Phe His LeuGlu Trp Leu Arg Pro Phe Phe Ser Ser Trp Leu 180 185 190 Val Leu Asn ValSer Trp Phe Leu Arg Arg Ser Pro Ala Asn His Val 195 200 205 Ser Val ArgVal Leu Gln Thr Leu Arg Pro Thr Pro Pro Gln Arg Gln 210 215 220 Ala LeuLeu Ser Ser Lys Thr Ser Val Ala Leu Gly Ile Ala Thr Arg 225 230 235 240Pro Leu Arg Arg Phe Ala Lys Ser Leu Ser Ala Val Arg Arg 245 250 19 254PRT Porcine reproductive and respiratory syndrome virus 19 Met Val AsnSer Cys Thr Phe Leu His Ile Phe Leu Cys Cys Ser Phe 1 5 10 15 Leu TyrSer Phe Cys Cys Ala Val Ala Ala Gly Ser Asn Ala Thr Tyr 20 25 30 Cys PheTrp Phe Pro Leu Val Arg Gly Asn Phe Ser Phe Glu Leu Met 35 40 45 Val AsnTyr Thr Val Cys Pro Pro Cys Leu Thr Arg Gln Ala Ala Ala 50 55 60 Glu ValTyr Glu Pro Gly Arg Ser Leu Trp Cys Arg Ile Gly Tyr Asp 65 70 75 80 ArgCys Gly Glu Asp Asp His Asp Glu Leu Gly Phe Met Ile Pro Pro 85 90 95 GlyLeu Ser Ser Glu Gly His Leu Thr Ser Val Tyr Ala Trp Leu Ala 100 105 110Phe Leu Ser Phe Ser Tyr Thr Ala Gln Phe His Pro Glu Ile Phe Gly 115 120125 Ile Gly Asn Val Ser Arg Val Tyr Val Asp Ile Lys His Gln Leu Ile 130135 140 Cys Ala Glu His Asp Gly Gln Asn Ala Thr Leu Pro Arg His Asp Asn145 150 155 160 Ile Ser Ala Val Phe Gln Thr Tyr Tyr Gln His Gln Val AspGly Gly 165 170 175 Asn Trp Phe His Leu Glu Trp Leu Arg Pro Phe Phe SerSer Trp Leu 180 185 190 Val Leu Asn Val Ser Trp Tyr Leu Arg Arg Ser ProAla Asn His Ala 195 200 205 Ser Val Arg Val Leu Gln Ile Leu Arg Pro ThrLeu Pro Gln Arg Gln 210 215 220 Ala Leu Leu Ser Ser Lys Thr Ser Val AlaLeu Gly Ile Ala Thr Arg 225 230 235 240 Pro Leu Arg Arg Phe Ala Lys SerLeu Ser Ala Val Arg Arg 245 250 20 254 PRT Porcine reproductive andrespiratory syndrome virus 20 Met Val Asn Ser Cys Thr Phe Leu His IlePhe Leu Cys Cys Ser Phe 1 5 10 15 Leu Tyr Ser Phe Cys Cys Ala Val ValAla Gly Ser Asn Thr Thr Phe 20 25 30 Cys Phe Trp Phe Pro Leu Val Arg GlyAsn Phe Ser Phe Glu Leu Thr 35 40 45 Val Asn Tyr Thr Val Cys Pro Pro CysLeu Thr Arg Gln Ala Ala Ala 50 55 60 Glu Ile Tyr Glu Pro Gly Arg Ser LeuTrp Cys Arg Ile Gly Tyr Asp 65 70 75 80 Arg Cys Gly Glu Asp Asp His AspGlu Leu Gly Phe Met Val Pro Pro 85 90 95 Gly Phe Ser Ser Glu Gly His LeuThr Ser Val Tyr Ala Trp Leu Ala 100 105 110 Phe Leu Ser Phe Ser Tyr ThrAla Gln Phe His Pro Glu Ile Phe Gly 115 120 125 Ile Gly Asn Val Ser ArgVal Tyr Val Asp Ile Lys His Gln Leu Ile 130 135 140 Cys Ala Glu His AspGly Gln Asn Thr Thr Leu Pro Arg His Asp Asn 145 150 155 160 Ile Ser AlaVal Phe Gln Thr Tyr Tyr Gln His Gln Val Asp Gly Gly 165 170 175 Asn TrpPhe His Leu Glu Trp Leu Arg Pro Phe Phe Ser Ser Trp Leu 180 185 190 ValLeu Asn Val Ser Trp Phe Leu Arg Arg Ser Pro Ala Asn His Val 195 200 205Ser Val Arg Val Leu Gln Ile Leu Arg Pro Thr Pro Pro Gln Arg Gln 210 215220 Ala Leu Leu Ser Ser Lys Thr Ser Val Ala Leu Gly Ile Ala Thr Arg 225230 235 240 Pro Leu Arg Arg Phe Ala Lys Ser Leu Ser Ala Val Arg Arg 245250 21 254 PRT Porcine reproductive and respiratory syndrome virus 21Met Ala Asn Ser Cys Thr Phe Leu His Ile Leu Leu Cys Cys Ser Phe 1 5 1015 Leu Tyr Ser Phe Cys Cys Val Val Val Thr Asp Ala Asn Ala Thr Phe 20 2530 Cys Phe Trp Phe Pro Leu Val Arg Gly Asn Phe Ser Phe Glu Leu Met 35 4045 Val Asn Tyr Thr Val Cys Pro Pro Cys Leu Thr Arg Gln Ala Ala Ala 50 5560 Gln Ile Tyr Glu Pro Asn Arg Ser Leu Trp Cys Arg Ile Gly Asn Asp 65 7075 80 Arg Cys Gly Glu Asp Asp His Asp Glu Leu Gly Phe Thr Val Pro Pro 8590 95 Gly Leu Ser Lys Glu Val His Leu Thr Ser Val Tyr Ala Trp Leu Ala100 105 110 Phe Leu Ser Phe Ser Tyr Thr Ala Gln Phe His Pro Glu Ile PheGly 115 120 125 Ile Gly Asn Val Ser Lys Val Tyr Val Asp Ile Asn His GlnLeu Ile 130 135 140 Cys Ala Val His Asp Gly Gln Asn Thr Thr Leu Pro ArgHis Asp Asn 145 150 155 160 Ile Ser Ala Val Phe Gln Thr Tyr Tyr Gln HisGln Val Asp Gly Gly 165 170 175 Asn Trp Phe His Leu Glu Trp Leu Arg ProPhe Phe Ser Ser Trp Leu 180 185 190 Val Leu Asn Val Ser Trp Phe Leu ArgArg Ser Pro Ala Ser His Val 195 200 205 Ser Val Arg Val Phe Gln Thr SerArg Pro Thr Pro Pro Arg Gln Gln 210 215 220 Ile Ser Leu Ser Ser Arg ThrSer Ala Ala Leu Gly Met Ala Thr Arg 225 230 235 240 Pro Leu Arg Arg PheAla Lys Ser Leu Ser Ala Ala Arg Arg 245 250 22 254 PRT Porcinereproductive and respiratory syndrome virus 22 Met Val Asn Ser Cys ThrPhe Leu His Ile Phe Leu Cys Cys Ser Phe 1 5 10 15 Leu Tyr Ser Phe CysCys Ala Val Val Ala Gly Ser Asn Thr Thr Tyr 20 25 30 Cys Phe Trp Phe ProLeu Val Arg Gly Asn Phe Ser Phe Glu Leu Thr 35 40 45 Val Asn Tyr Thr ValCys Pro Pro Cys Leu Thr Arg Gln Ala Ala Thr 50 55 60 Glu Ile Tyr Glu ProGly Arg Ser Leu Trp Cys Arg Ile Gly Tyr Asp 65 70 75 80 Arg Cys Gly GluAsp Asp His Asp Glu Leu Gly Phe Met Ile Pro Pro 85 90 95 Gly Leu Ser SerGlu Gly His Leu Thr Gly Val Tyr Ala Trp Leu Ala 100 105 110 Phe Leu SerPhe Ser Tyr Thr Ala Gln Phe His Pro Glu Ile Phe Gly 115 120 125 Ile GlyAsn Val Ser Arg Val Tyr Val Asp Ile Lys His Gln Leu Ile 130 135 140 CysAla Glu His Asp Gly Gln Asn Thr Thr Leu Pro Arg His Asp Asn 145 150 155160 Ile Ser Ala Val Phe Gln Thr Tyr Tyr Gln His Gln Val Asp Gly Gly 165170 175 Asn Trp Phe His Leu Glu Trp Leu Arg Pro Phe Phe Ser Ser Trp Leu180 185 190 Val Leu Asn Val Ser Trp Phe Leu Arg Arg Ser Pro Ala Asn HisVal 195 200 205 Ser Val Arg Val Leu Gln Ile Leu Arg Pro Thr Pro Pro GlnArg Gln 210 215 220 Ala Leu Leu Ser Ser Lys Thr Ser Val Ala Leu Gly IleAla Thr Arg 225 230 235 240 Pro Leu Arg Arg Phe Ala Lys Ser Leu Ser AlaVal Arg Arg 245 250 23 265 PRT Porcine reproductive and respiratorysyndrome virus 23 Met Ala His Gln Cys Ala Arg Phe His Phe Phe Leu CysGly Phe Ile 1 5 10 15 Cys Tyr Leu Val His Ser Ala Leu Ala Ser Asn SerSer Ser Thr Leu 20 25 30 Cys Phe Trp Phe Pro Leu Ala His Gly Asn Thr SerPhe Glu Leu Thr 35 40 45 Ile Asn Tyr Thr Ile Cys Met Pro Cys Ser Thr SerGln Ala Ala Arg 50 55 60 Gln Arg Leu Glu Pro Gly Arg Asn Met Trp Cys LysIle Gly His Asp 65 70 75 80 Arg Cys Glu Glu Arg Asp His Asp Glu Leu LeuMet Ser Ile Pro Ser 85 90 95 Gly Tyr Asp Asn Leu Lys Leu Glu Gly Tyr TyrAla Trp Leu Ala Phe 100 105 110 Leu Ser Phe Ser Tyr Ala Ala Gln Phe HisPro Glu Leu Phe Gly Ile 115 120 125 Gly Asn Val Ser Arg Val Phe Val AspLys Arg His Gln Phe Ile Cys 130 135 140 Ala Glu His Asp Gly His Asn SerThr Val Ser Thr Gly His Asn Ile 145 150 155 160 Ser Ala Leu Tyr Ala AlaTyr Tyr His His Gln Ile Asp Gly Gly Asn 165 170 175 Trp Phe His Leu GluTrp Leu Arg Pro Leu Phe Ser Ser Trp Leu Val 180 185 190 Leu Asn Ile SerTrp Phe Leu Arg Arg Ser Pro Val Ser Pro Val Ser 195 200 205 Arg Arg IleTyr Gln Ile Leu Arg Pro Thr Arg Pro Arg Leu Pro Val 210 215 220 Ser TrpSer Phe Arg Thr Ser Ile Val Ser Asp Leu Thr Gly Ser Gln 225 230 235 240Gln Arg Lys Arg Lys Phe Pro Ser Glu Ser Arg Pro Asn Val Val Lys 245 250255 Pro Ser Val Leu Pro Ser Thr Ser Arg 260 265 24 178 PRT Porcinereproductive and respiratory syndrome virus 24 Met Gly Ala Ser Leu LeuPhe Leu Leu Val Gly Phe Lys Cys Leu Leu 1 5 10 15 Val Ser Gln Ala PheAla Cys Lys Pro Cys Phe Ser Ser Ser Leu Ser 20 25 30 Asp Ile Lys Thr AsnThr Thr Ala Ala Ala Gly Phe Ala Val Leu Gln 35 40 45 Asp Ile Ser Cys LeuArg His Arg Asn Ser Ala Ser Glu Ala Ile Arg 50 55 60 Lys Val Pro Gln CysArg Thr Ala Ile Gly Thr Pro Val Tyr Ile Thr 65 70 75 80 Val Thr Ala AsnVal Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu 85 90 95 Leu Met Leu SerSer Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys 100 105 110 Gly Phe LysVal Val Phe Gly Asn Val Ser Gly Ile Val Ala Val Cys 115 120 125 Val AsnPhe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg 130 135 140 SerLeu Val Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu 145 150 155160 Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Thr Ile Leu Leu 165170 175 Ala Ile 25 178 PRT Porcine reproductive and respiratory syndromevirus 25 Met Ala Ser Ser Leu Leu Phe Leu Val Val Gly Phe Lys Cys Leu Leu1 5 10 15 Val Ser Gln Ala Phe Ala Cys Lys Pro Cys Phe Ser Ser Ser LeuAla 20 25 30 Asp Ile Lys Thr Asn Thr Thr Ala Ala Ala Ser Phe Ala Val LeuGln 35 40 45 Asp Ile Ser Cys Leu Arg His Arg Asp Ser Ala Ser Glu Ala IleArg 50 55 60 Lys Ile Pro Gln Cys Arg Thr Ala Ile Gly Thr Pro Val Tyr ValThr 65 70 75 80 Ile Thr Ala Asn Val Thr Asp Glu Asn Tyr Leu His Ser SerAsp Leu 85 90 95 Leu Met Leu Ser Ser Cys Leu Phe Tyr Ala Ser Glu Met SerGlu Lys 100 105 110 Gly Phe Lys Val Val Phe Gly Asn Val Ser Gly Ile ValAla Val Cys 115 120 125 Val Asn Phe Thr Ser Tyr Val Gln His Val Lys GluPhe Thr Gln Arg 130 135 140 Ser Leu Val Val Asp His Val Arg Leu Leu HisPhe Met Thr Pro Glu 145 150 155 160 Thr Met Arg Trp Ala Thr Val Leu AlaCys Leu Phe Ala Ile Leu Leu 165 170 175 Ala Ile 26 178 PRT Porcinereproductive and respiratory syndrome virus 26 Met Ala Ala Ser Leu LeuPhe Leu Leu Val Gly Phe Lys Cys Leu Leu 1 5 10 15 Val Ser Gln Ala PheAla Cys Lys Pro Cys Phe Ser Ser Ser Leu Ala 20 25 30 Asp Ile Lys Thr AsnThr Thr Ala Ala Ala Gly Phe Ala Val Leu Gln 35 40 45 Asp Ile Ser Cys LeuArg Tyr Arg Asn Ser Ala Ser Glu Ala Phe Arg 50 55 60 Lys Ile Pro Gln CysArg Thr Ala Ile Gly Thr Pro Met Tyr Ile Thr 65 70 75 80 Val Thr Ala AsnVal Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu 85 90 95 Leu Met Leu SerSer Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys 100 105 110 Gly Phe LysVal Val Phe Gly Asn Val Ser Gly Ile Val Ala Val Cys 115 120 125 Val AsnPhe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg 130 135 140 SerLeu Val Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu 145 150 155160 Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu 165170 175 Ala Ile 27 178 PRT Porcine reproductive and respiratory syndromevirus 27 Met Ala Ser Ser Leu Leu Phe Leu Met Val Gly Phe Lys Cys Leu Leu1 5 10 15 Val Ser Gln Ala Phe Ala Cys Lys Pro Cys Phe Ser Ser Ser LeuAla 20 25 30 Asp Ile Lys Thr Asn Thr Thr Ala Ala Ala Ser Phe Ala Val LeuGln 35 40 45 Asp Ile Ser Cys Leu Arg His Arg Asn Ser Ala Ser Glu Ala IleArg 50 55 60 Lys Ile Pro Gln Cys Arg Thr Ala Ile Gly Thr Pro Val Tyr IleThr 65 70 75 80 Ile Thr Ala Asn Val Thr Asp Glu Asn Tyr Leu His Ser SerAsp Leu 85 90 95 Leu Met Leu Ser Ser Cys Leu Phe Tyr Ala Ser Glu Met SerGlu Lys 100 105 110 Gly Phe Lys Val Val Phe Gly Asn Val Ser Gly Ile ValAla Val Cys 115 120 125 Val Asn Phe Thr Ser Tyr Val Gln His Val Arg GluPhe Thr Gln Arg 130 135 140 Ser Leu Val Val Asp His Val Arg Leu Leu HisPhe Met Thr Pro Glu 145 150 155 160 Thr Met Arg Trp Ala Thr Val Leu AlaCys Leu Phe Ala Ile Leu Leu 165 170 175 Ala Ile 28 178 PRT Porcinereproductive and respiratory syndrome virus 28 Met Ala Ser Ser Leu LeuPhe Leu Met Val Gly Phe Lys Cys Leu Leu 1 5 10 15 Val Ser Gln Ala PheAla Cys Lys Pro Cys Phe Ser Ser Ser Leu Ala 20 25 30 Asp Ile Lys Thr AsnThr Thr Ala Ala Ala Ser Phe Ala Val Leu Gln 35 40 45 Asp Ile Gly Cys LeuArg His Arg Asp Ser Ala Ser Glu Ala Ile Arg 50 55 60 Lys Ile Pro Gln CysArg Thr Ala Ile Gly Thr Pro Val Tyr Ile Thr 65 70 75 80 Ile Thr Ala AsnVal Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu 85 90 95 Leu Met Leu SerSer Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys 100 105 110 Gly Phe LysVal Val Phe Gly Asn Val Ser Gly Ile Val Ala Val Cys 115 120 125 Val AsnPhe Thr Ser Tyr Val Gln His Val Arg Glu Phe Thr Gln Arg 130 135 140 SerLeu Val Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu 145 150 155160 Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu 165170 175 Ala Ile 29 178 PRT Porcine reproductive and respiratory syndromevirus 29 Met Ala Ala Ser Leu Leu Phe Leu Met Val Gly Phe Lys Cys Leu Leu1 5 10 15 Val Ser Gln Ala Phe Ala Cys Lys Pro Cys Phe Ser Ser Ser LeuAla 20 25 30 Asp Ile Lys Thr Asn Thr Thr Ala Ala Ala Ser Phe Ala Val LeuGln 35 40 45 Asp Ile Ser Cys Leu Arg His Arg Asn Ser Ala Ser Glu Ala IleArg 50 55 60 Lys Ile Pro Gln Cys Arg Thr Ala Ile Gly Thr Pro Met Tyr IleThr 65 70 75 80 Ile Thr Ala Asn Val Thr Asp Glu Asn Tyr Leu His Ser SerAsp Leu 85 90 95 Leu Met Leu Ser Ser Cys Leu Phe Tyr Ala Ser Glu Met SerGlu Lys 100 105 110 Gly Phe Glu Val Val Phe Gly Asn Val Ser Gly Ile ValAla Val Cys 115 120 125 Val Asn Phe Thr Ser Tyr Val Gln His Val Arg GluPhe Thr Gln Arg 130 135 140 Ser Leu Met Val Asp His Val Arg Leu Leu HisPhe Met Thr Pro Glu 145 150 155 160 Thr Met Arg Trp Ala Thr Val Leu AlaCys Leu Phe Ala Ile Leu Leu 165 170 175 Ala Ile 30 178 PRT Porcinereproductive and respiratory syndrome virus 30 Met Ala Ala Ser Leu LeuPhe Leu Leu Val Gly Phe Glu Cys Leu Leu 1 5 10 15 Val Ser Gln Ala PheAla Cys Lys Pro Cys Phe Ser Ser Ser Leu Ser 20 25 30 Asp Ile Lys Thr AsnThr Thr Ala Ala Ala Asn Phe Ala Val Leu Gln 35 40 45 Asp Ile Gly Cys LeuArg His Gly Asn Ser Thr Thr Glu Ala Phe Arg 50 55 60 Lys Ile Pro Gln CysArg Thr Ala Ile Gly Thr Pro Val Tyr Ile Thr 65 70 75 80 Ile Thr Ala AsnVal Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu 85 90 95 Leu Met Leu SerSer Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys 100 105 110 Gly Phe LysVal Val Phe Gly Asn Val Ser Gly Thr Val Ala Val Cys 115 120 125 Ile AsnPhe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg 130 135 140 SerLeu Val Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu 145 150 155160 Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu 165170 175 Ala Ile 31 183 PRT Porcine reproductive and respiratory syndromevirus 31 Met Ala Ala Ala Thr Leu Phe Phe Leu Ala Gly Ala Gln His Ile Met1 5 10 15 Val Ser Glu Ala Phe Ala Cys Lys Pro Cys Phe Ser Thr His LeuSer 20 25 30 Asp Ile Glu Thr Asn Thr Thr Ala Ala Ala Gly Phe Met Val LeuGln 35 40 45 Asp Ile Asn Cys Phe Arg Pro His Gly Val Ser Ala Ala Gln GluLys 50 55 60 Ile Ser Phe Gly Lys Ser Ser Gln Cys Arg Glu Ala Val Gly ThrPro 65 70 75 80 Gln Tyr Ile Thr Ile Thr Ala Asn Val Thr Asp Glu Ser TyrLeu Tyr 85 90 95 Asn Ala Asp Leu Leu Met Leu Ser Ala Cys Leu Phe Tyr AlaSer Glu 100 105 110 Met Ser Glu Lys Gly Phe Lys Val Ile Phe Gly Asn ValSer Gly Val 115 120 125 Val Ser Ala Cys Val Asn Phe Thr Asp Tyr Val AlaHis Val Thr Gln 130 135 140 His Thr Gln Gln His His Leu Val Ile Asp HisIle Arg Leu Leu His 145 150 155 160 Phe Leu Thr Pro Ser Ala Met Arg TrpAla Thr Thr Ile Ala Cys Leu 165 170 175 Phe Ala Ile Leu Leu Ala Ile 18032 199 PRT Porcine reproductive and respiratory syndrome virus 32 MetLeu Gly Lys Cys Leu Thr Ala Gly Cys Cys Ser Gln Leu Leu Phe 1 5 10 15Leu Trp Cys Ile Val Pro Ser Cys Phe Val Ala Leu Val Ser Ala Asn 20 25 30Gly Asn Ser Gly Ser Asn Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys 35 40 45Glu Leu Asn Gly Thr Asp Trp Leu Ala Asn Lys Phe Asp Trp Ala Val 50 55 60Glu Cys Phe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly 65 70 7580 Ala Leu Thr Thr Ser His Phe Leu Asp Thr Val Gly Leu Val Thr Val 85 9095 Ser Thr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Met Tyr 100105 110 Ala Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala115 120 125 Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr AsnPhe 130 135 140 Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser ProVal Ile 145 150 155 160 Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly HisLeu Ile Asp Leu 165 170 175 Lys Arg Val Val Leu Asp Gly Ser Ala Ala ThrPro Val Thr Arg Val 180 185 190 Ser Ala Glu Gln Trp Arg Pro 195 33 199PRT Porcine reproductive and respiratory syndrome virus 33 Met Leu GluLys Cys Leu Thr Ala Gly Cys Cys Ser Arg Leu Leu Ser 1 5 10 15 Leu TrpCys Ile Val Pro Phe Cys Phe Ala Val Leu Ala Asn Ala Ser 20 25 30 Asn AspSer Ser Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys 35 40 45 Glu LeuAsn Gly Thr Asp Trp Leu Ala Asn Lys Phe Asp Trp Ala Val 50 55 60 Glu SerPhe Val Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly 65 70 75 80 AlaLeu Thr Thr Ser His Phe Leu Asp Thr Val Ala Leu Val Thr Val 85 90 95 SerThr Ala Gly Phe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr 100 105 110Ala Val Cys Ala Leu Ala Ala Leu Thr Cys Phe Val Ile Arg Phe Ala 115 120125 Lys Asn Cys Met Ser Trp Arg Tyr Ala Cys Thr Arg Tyr Thr Asn Phe 130135 140 Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile145 150 155 160 Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu IleAsp Leu 165 170 175 Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro IleThr Arg Val 180 185 190 Ser Ala Glu Gln Gly Arg Pro 195 34 199 PRTPorcine reproductive and respiratory syndrome virus 34 Met Leu Gly LysCys Leu Thr Ala Gly Tyr Cys Ser Ser Leu Leu Phe 1 5 10 15 Leu Trp CysIle Val Pro Ser Trp Phe Val Ala Leu Ala Ser Ala Asn 20 25 30 Ser Ser AsnSer Ser His Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys 35 40 45 Glu Leu AsnGly Thr Asp Trp Leu Ala Gly Glu Phe Asp Trp Ala Val 50 55 60 Glu Cys PheVal Ile Phe Pro Val Leu Thr His Ile Val Ser Tyr Gly 65 70 75 80 Ala LeuThr Thr Ser His Phe Leu Asp Thr Val Gly Leu Val Thr Val 85 90 95 Ser ThrAla Gly Phe Ser His Gly Arg Tyr Val Leu Ser Ser Ile Tyr 100 105 110 AlaVal Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Phe Thr 115 120 125Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe 130 135140 Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile 145150 155 160 Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile AspLeu 165 170 175 Lys Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro Ile ThrLys Val 180 185 190 Ser Ala Glu Gln Gly Arg Pro 195 35 199 PRT Porcinereproductive and respiratory syndrome virus 35 Met Leu Gly Lys Cys LeuThr Ala Gly Cys Cys Ser Arg Leu Leu Ser 1 5 10 15 Leu Trp Cys Ile ValPro Phe Cys Phe Ala Val Leu Ala Asn Ala Ser 20 25 30 Ala Asn Ser Ser SerHis Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys 35 40 45 Glu Leu Asn Gly ThrAsp Trp Leu Ala Asp Lys Phe Asp Trp Ala Val 50 55 60 Glu Ser Phe Val IlePhe Pro Val Leu Thr His Ile Val Ser Tyr Gly 65 70 75 80 Ala Leu Thr ThrSer His Leu Leu Asp Thr Val Ala Leu Val Thr Val 85 90 95 Ser Thr Ala GlyPhe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr 100 105 110 Ala Val CysAla Leu Ala Ala Leu Ala Cys Phe Val Ile Arg Phe Ala 115 120 125 Lys AsnCys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe 130 135 140 LeuLeu Asp Thr Lys Gly Arg Leu Tyr Arg Trp His Ser Pro Val Ile 145 150 155160 Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu 165170 175 Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val180 185 190 Ser Ala Glu Gln Gly Arg Pro 195 36 199 PRT Porcinereproductive and respiratory syndrome virus 36 Met Leu Gly Lys Cys LeuThr Val Gly Cys Cys Ser Arg Leu Leu Ser 1 5 10 15 Leu Trp Cys Ile ValPro Phe Cys Phe Thr Val Leu Ala Asp Ala His 20 25 30 Ser Asn Ser Ser SerHis Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys 35 40 45 Glu Leu Asn Gly ThrAsp Trp Leu Ala Asp Arg Phe Asp Trp Ala Val 50 55 60 Glu Ser Phe Val IlePhe Pro Val Leu Thr His Ile Val Ser Tyr Gly 65 70 75 80 Ala Leu Thr ThrSer His Phe Leu Asp Thr Ile Ala Leu Val Thr Val 85 90 95 Ser Thr Ala GlyPhe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr 100 105 110 Ala Val CysAla Leu Ala Ala Leu Thr Cys Phe Val Ile Arg Phe Val 115 120 125 Lys AsnCys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe 130 135 140 LeuLeu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile 145 150 155160 Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu 165170 175 Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val180 185 190 Ser Ala Glu Gln Gly Arg Pro 195 37 199 PRT Porcinereproductive and respiratory syndrome virus 37 Met Leu Gly Lys Cys LeuThr Ala Gly Cys Cys Ser Arg Leu Leu Ser 1 5 10 15 Leu Trp Phe Ile ValPro Phe Cys Phe Ala Val Leu Ala Ser Ala Ser 20 25 30 Asn Ser Ser Ser SerHis Leu Gln Leu Ile Tyr Asn Leu Thr Leu Cys 35 40 45 Glu Leu Asn Gly ThrAsp Trp Leu Ala Asn Lys Phe Asp Trp Ala Val 50 55 60 Glu Ser Phe Val IlePhe Pro Val Leu Thr His Ile Val Ser Tyr Gly 65 70 75 80 Ala Leu Thr ThrSer His Phe Leu Asp Thr Val Ala Leu Val Thr Val 85 90 95 Ser Thr Ala GlyPhe Val His Gly Arg Tyr Val Leu Ser Ser Ile Tyr 100 105 110 Ala Val CysAla Leu Ala Ala Leu Thr Cys Phe Ile Ile Arg Phe Ala 115 120 125 Lys AsnCys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe 130 135 140 LeuLeu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile 145 150 155160 Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu 165170 175 Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro Ile Thr Arg Val180 185 190 Ser Ala Glu Gln Gly Arg Pro 195 38 198 PRT Porcinereproductive and respiratory syndrome virus 38 Met Leu Gly Lys Cys LeuThr Ala Gly Cys Cys Ser Arg Ser Leu Phe 1 5 10 15 Leu Trp Cys Ile ValPro Phe Cys Leu Ala Ala Leu Val Ser Ala Asn 20 25 30 Asn Ser Ser Ser HisLeu Gln Leu Ile Tyr Asn Leu Thr Leu Cys Glu 35 40 45 Leu Asn Gly Thr AspTrp Leu Ala Asn Lys Phe Asp Trp Ala Val Glu 50 55 60 Ser Phe Val Ile PhePro Val Leu Thr His Ile Val Ser Tyr Gly Ala 65 70 75 80 Leu Thr Thr SerHis Phe Leu Asp Thr Val Gly Leu Val Thr Val Ser 85 90 95 Thr Ala Gly PheHis His Gly Arg Tyr Val Leu Ser Ser Ile Tyr Ala 100 105 110 Val Cys AlaLeu Ala Ala Phe Ile Cys Phe Val Ile Arg Phe Ala Lys 115 120 125 Asn CysMet Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe Leu 130 135 140 LeuAsp Thr Lys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile Ile 145 150 155160 Glu Lys Gly Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu Lys 165170 175 Lys Val Val Leu Asp Gly Ser Ala Ala Thr Pro Leu Thr Arg Val Ser180 185 190 Ala Glu Gln Gly Arg Pro 195 39 200 PRT Porcine reproductiveand respiratory syndrome virus 39 Met Arg Cys Ser His Lys Leu Gly ArgPhe Leu Thr Pro His Ser Cys 1 5 10 15 Phe Trp Trp Leu Phe Leu Leu CysThr Gly Leu Ser Trp Ser Phe Ser 20 25 30 Asp Asn Gly Gly Asp Ser Ser ThrTyr Gln Tyr Ile Tyr Asn Leu Thr 35 40 45 Ile Cys Glu Leu Asn Gly Thr AspTrp Leu Ser Ser His Phe Gly Trp 50 55 60 Ala Val Glu Thr Phe Val Leu TyrPro Val Ala Thr His Ile Leu Ser 65 70 75 80 Leu Gly Phe Leu Thr Thr SerHis Phe Phe Asp Ala Leu Gly Leu Gly 85 90 95 Ala Val Ser Thr Ala Gly PheVal Gly Gly Arg Tyr Val Leu Cys Ser 100 105 110 Val Tyr Gly Ala Cys AlaPhe Ala Ala Phe Val Cys Phe Val Ile Arg 115 120 125 Ala Ala Lys Asn CysMet Ala Cys Arg Tyr Ala Arg Thr Arg Phe Thr 130 135 140 Asn Phe Ile ValAsp Asp Arg Gly Arg Val His Arg Trp Lys Ser Pro 145 150 155 160 Ile ValVal Glu Lys Leu Gly Lys Ala Glu Val Asp Gly Asn Leu Val 165 170 175 ThrIle Lys His Val Val Leu Glu Gly Val Lys Ala Gln Pro Leu Thr 180 185 190Arg Thr Ser Ala Glu Gln Glu Ala 195 200 40 3293 DNA Porcine reproductiveand respiratory syndrome virus 40 gttttatttc cctccgggcc ctgtcattgaaccaacttta ggcctgaatt gaaatgaaat 60 ggggtccatg caaagccttt ttgacaaaattggccaactt tttgtggatg ctttcacgga 120 gttcttggtg tccattgttg atatcattatattcttggcc attttgtttg gcttcaccat 180 cgccggttgg ctggtggtct tttgcatcagattggtttgc tccgcgatac tccgtacgcg 240 ccctgccatt cactctgagc aattacagaagatcttatga ggcctttctt tcccagtgcc 300 aagtggacat tcccacctgg ggaactaaacatcctttggg gatgttttgg caccataagg 360 tgtcaaccct gattgatgag atggtgtcgcgtcgaatgta ccgcatcatg gaaaaagcag 420 gacaggctgc ctggaaacag gtggtgagcgaggctacgct gtctcgcatt agtagtttgg 480 atgtggtggc tcattttcag catcttgccgccatcgaagc cgagacctgt aaatatttgg 540 cctcccggct gcccatgcta cacaacctgcgcatgacagg gtcaaatgta accatagtgt 600 ataatagtac tttgaatcgg gtgtttgctattttcccaac ccctggttcc cggccaaagc 660 ttcatgactt tcagcaatgg ctaatagctgtgcattcctc catattttcc tctgttgcag 720 cttcttgtac tctctttgtt gtgctgtggttgcgggttcc aatactacgt actgtttttg 780 gtttccgctg gttaggggca atttttctttcgaactcata gtgaattaca cggtgtgccc 840 accttgcctc acccggcaag cagccgcagaggcctacgaa cccggtaggt ctctttggtg 900 caggataggg tacgatcgat gtggagaggacgaccatgac gagctagggt ttatgatacc 960 gtctggcctc tccagcgaag gccacttgaccagtgtttac gcctggttgg cgttcttgtc 1020 cttcagctac acggcccagt tccaccccgagatattcggg atagggaatg tgagtcgagt 1080 ttatgttgac atcaaacatc aactcatctgcgccgaacat gacgggcaga acaccacctt 1140 gcctcgtcat gacaacattt cggccgtgtttcagacctat taccaacatc aagtcgacgg 1200 cggcaattgg tttcacctag aatggctgcgtcccttcttt tcctcatggt tggttttaaa 1260 tgtctcttgg tttctcaggc gttcgcctgcaaaccatgtt tcagttcgag tcttgcagac 1320 attaagacca acaccaccgc agcggcaagctttgctgtcc tccaagacat cagttgcctt 1380 aggcatcgca actcggcctc tgaggcgattcgcaaaatcc ctcagtgccg tacggcgata 1440 gggacaccta tgtatattac catcacagccaatgtgacag atgaaaatta tttacattct 1500 tctgatctcc tcatgctctc ttcttgccttttctatgctt ctgagatgag tgaaaaggga 1560 tttgaggtgg tttttggcaa tgtgtcaggcatcgtggctg tgtgtgtcaa ttttaccagc 1620 tacgttcaac atgtcaggga gtttacccaacgctccttga tggtcgacca tgtgcggctg 1680 ctccatttca tgacacctga gaccatgaggtgggcaaccg ttttagcctg tctttttgct 1740 attctgttgg caatttgaat gtttaagtatgttggggaaa tgcttgaccg tgggctgttg 1800 ctcgcgattg ctttctttgt ggtgtatcgtgccgttctgt tttactgtgc tcgccgacgc 1860 ccacagcaac agcagctctc atctgcaattgatttacaac ttgacgctat gtgagctgaa 1920 tggcacagat tggctagctg atagatttgattgggcagtg gagagctttg tcatctttcc 1980 tgttttgact cacattgtct cctatggcgccctcaccacc agccatttcc ttgacacaat 2040 tgctttagtc actgtgtcta ccgccgggtttgttcacggg cggtatgtcc taagtagcat 2100 ctacgcggtc tgtgccctgg ctgcgttgacttgcttcgtc attaggtttg tgaagaattg 2160 catgtcctgg cgctactcat gtactagatataccaacttt cttctggata ctaagggcag 2220 actctatcgt tggcggtcgc ctgtcatcatagagaagagg ggcaaagttg aggtcgaagg 2280 tcatctgatc gatctcaaaa gagttgtgcttgatggttcc gtggcaaccc ctataaccag 2340 agtttcagcg gaacaatggg gtcgtccttagatgacttct gttatgatag tacggctcca 2400 caaaaggtgc ttttggcatt ttctattacctacacgccag taatgatata tgccctaaag 2460 gtgagtcgcg gccgactgct agggcttctgcaccttttga ttttcctgaa ctgtgctttc 2520 accttcgggt acatgacatt catgcactttcagagtacaa ataaggtcgc gctcactatg 2580 ggagcagtag ttgcactcct ttggggggtgtactcagcca tagaaacctg gaaattcatc 2640 acctccagat gccgtttgtg cttgctaggccgcaagtaca ttctggcccc tgcccaccac 2700 gttgaaagtg ccgcaggctt tcatccgattgcggcaaatg ataaccacgc atttgtcgtc 2760 cggcgtcccg gctccactac ggtcaacggcacattggtgc ccgggttgaa aagcctcgtg 2820 ttgggtggca gaaaagctgt taaacagggagtggtaaacc ttgtcaaata tgccaaataa 2880 caacggcaag cagcagaaga gaaagaagggggatggccag ccagtcaatc agctgtgcca 2940 gatgctgggt aagatcatcg cccagcaaaaccagtctaga ggcaagggac cgggaaagaa 3000 aaataagaag aaaaacccgg agaagccccattttcctcta gcgactgaag atgatgtcag 3060 acatcacttt acccctagtg agcggcaattgtgtctgtcg tcaatccaaa ctgcctttaa 3120 tcaaggcgct gggacttgca ccctgtcagattcagggagg ataagttaca ctgtggagtt 3180 tagtttgcct acgcatcata ctgtgcgcttgatccgcgtc acagcatcac cctcagcatg 3240 atgggctggc attcttgagg catcccagtgtttgaattgg aagaatgcgt ggt 3293 41 3293 DNA Porcine reproductive andrespiratory syndrome virus 41 gttttatttc cctccgggcc ccgtcattgaaccaacttta ggcctgaatt gaaatgaaat 60 ggggtccgtg caaagccttt ttgacaaaattggccaactt tttgtggatg ctttcacgga 120 gttcctggtg tccattgttg atatcatcatatttttggcc attttgtttg gcttcaccat 180 cgccggttgg ctggtggtct tttgcatcagattggtttgc tccgcgatac tccgtacgcg 240 ccctgccatt cactctgagc aattacagaagatcttatga ggccttttta tcccagtgcc 300 aagtggacat tcccacctgg ggaactaaacatcctttggg gatgttttgg caccataagg 360 tgtcaaccct gattgatgaa atggtgtcgcgtcgcatgta ccgcatcatg gaaaaagcag 420 ggcaggctgc ctggaaacag gtggtgagcgaggctacgct gtcccgcatt agtagtttgg 480 atgtggtggc tcattttcag catcttgccgccattgaagc cgagacttgt aaatatttgg 540 cctcccggct gcccatgcta cataacctgcgcataacagg gtcaaatgta accatagtgt 600 ataatagtac ttcggagcag gtgtttgctattttcccaac ccctggttcc cggccaaagc 660 ttcatgattt tcagcaatgg ttaatagctgtacattcctc catattttcc tctgttgcag 720 cttcttgtac tctttttgtt gtgctgtggctgcgggttcc aatgctacgt actgtttttg 780 gtttccgctg gttaggggga atttttccttcgaactcatg gtgaattaca cggtgtgtcc 840 accttgcctc acccggcaag cagccgcagaggtctacgaa cccggtaggt ctctttggtg 900 caggataggg tatgaccgat gtggggaggacgatcatgac gagctagggt ttatgatacc 960 gcctggcctc tccagcgaag gccacttgactagtgtttac gcctggttgg cgtttttgtc 1020 cttcagctac acggcccagt tccatcccgagatattcggg atagggaatg tgagtcgagt 1080 ttatgttgac atcaaacatc aactcatttgcgccgaacat gacggacaga acgccacctt 1140 gcctcgtcat gacaatattt cagccgtgtttcagacctat taccaacatc aagtcgatgg 1200 cggcaattgg tttcacctag aatggcttcgtcccttcttt tcctcatggt tggttttaaa 1260 tgtctcttgg tatctcaggc gttcgcctgcaaaccatgct tcagttcgag tcttgcagat 1320 attaagacca acactaccgc agcggcaagctttgctgtcc tccaagacat cagttgcctt 1380 aggcatcgca actcggcctc tgaggcgattcgcaaaatcc ctcagtgccg tacggcgata 1440 gggacacccg tgtatattac catcacagccaatgtgacag atgagaatta tttacattct 1500 tctgatctcc tcatgctttc ttcttgccttttctacgctt ctgagatgag tgaaaaagga 1560 ttcaaggtgg tatttggcaa tgtgtcaggcatcgtggctg tgtgtgtcaa ttttaccagc 1620 tacgtccaac atgtcaggga gtttacccaacgctccctgg tggtcgacca tgtgcggttg 1680 ctccatttca tgacacctga aaccatgaggtgggcaactg ttttagcctg tctttttgcc 1740 attctgctgg caatttgaat gtttaagtatgttggggaaa tgcttgaccg cgggctgttg 1800 ctcgcgattg ctttctttgt ggtgtatcgtgccgttctgt tttgctgtgc tcgccaacgc 1860 cagcgccaac agcagctccc atctacagctgatttacaac ttgacgctat gtgagctgaa 1920 tggcacagat tggctagctg ataaatttgattgggcagtg gagagttttg tcatctttcc 1980 cgttttgact cacattgtct cctatggtgccctcactact agccatctcc ttgacacagt 2040 cgccttagtc actgtgtcta ccgccgggtttgttcacggg cggtatgtcc taagtagcat 2100 ctacgcggtc tgtgccctgg ctgcgttagcttgcttcgtc attaggtttg caaagaattg 2160 catgtcctgg cgctattcgt gtaccagatataccaacttt cttctggaca ctaagggcag 2220 actctatcgt tggcattcgc ctgtcatcatagagaaaagg ggcaaagttg aggtcgaagg 2280 tcatctgatc gacctcaaaa gagttgtgcttgacggttcc gtggcaaccc ctataaccag 2340 agtttcagcg gaacaatggg gtcgtccttagatgacttct gccatgatag tacggctcca 2400 caaaaggtgc ttttggcgtt ttctattacctacacgccag tgatgatata tgccctaaag 2460 gtgagtcgcg gccgactgct agggcttctgcaccttttga tcttcctgaa ttgtgctttc 2520 accttcgggt acatgacatt cgtgcactttcagagtacaa ataaggtcgc gctcactatg 2580 ggagcagtag ttgcactcct ttggggggtgtactcagcca tagaaacctg gaaattcatc 2640 acctccagat gccgtttgtg cttgctaggccgcaagtaca ttctggcccc tgcccaccac 2700 gttgaaagtg ccgcaggctt tcatccgattgcggcaaatg ataaccacgc atttgtcgtc 2760 cggcgtcccg gctccactac ggtcaacggcacattggtgc ccgggttgaa aagcctcgtg 2820 ttgggtggca gaaaagctgt taaacagggagtggtaaacc ttgtcaaata tgccaaataa 2880 caacggcaag cagcagaaga gaaagaagggggatggccag ccagtcaatc agctgtgcca 2940 gatgctgggt aagatcatcg ctcagcaaaaccagtccaga ggcaagggac cgggaaagaa 3000 aaacaagaag aaaaacccgg agaagccccattttcctcta gcgactgaag atgatgtcag 3060 acatcacttc acccctagtg agcggcaattgtgtctgtcg tcaatccaga ccgcctttaa 3120 tcaaggcgct gggacttgca ccctgtcagattcagggagg ataagttaca ctgtggagtt 3180 tagtttgcca acgcatcata ctgtgcgcttgatccgcgtc acagcatcac cctcagcatg 3240 atgggctggc attcttgagg catcccagtgtttgaattgg aagaatgcgt ggt 3293 42 5 PRT Artificial Sequence Descriptionof Artificial SequencePeptide 42 Pro Ser Ser Ser Trp 1 5 43 5 PRTArtificial Sequence Description of Artificial SequencePeptide 43 Arg GlnArg Ile Ser 1 5 44 4 PRT Artificial Sequence Description of ArtificialSequencePeptide 44 Phe Gln Thr Ser 1 45 7 PRT Artificial SequenceDescription of Artificial SequencePeptide 45 Asn Gly Asn Ser Gly Ser Asn1 5 46 7 PRT Artificial Sequence Description of ArtificialSequencePeptide 46 Ser Asn Asp Ser Ser Ser His 1 5 47 7 PRT ArtificialSequence Description of Artificial SequencePeptide 47 Ser Ser Ser AsnSer Ser His 1 5 48 7 PRT Artificial Sequence Description of ArtificialSequencePeptide 48 Ser Ala Asn Ser Ser Ser His 1 5 49 7 PRT ArtificialSequence Description of Artificial SequencePeptide 49 His Ser Asn SerSer Ser His 1 5 50 7 PRT Artificial Sequence Description of ArtificialSequencePeptide 50 Ser Asn Ser Ser Ser Ser His 1 5 51 7 PRT ArtificialSequence Description of Artificial SequencePeptide 51 Asn Asn Ser SerSer Ser His 1 5 52 7 PRT Artificial Sequence Description of ArtificialSequencePeptide 52 Asn Gly Gly Asp Ser Ser Thr 1 5 53 7 PRT ArtificialSequence Description of Artificial SequencePeptide 53 Asn Gly Gly AspSer Ser Tyr 1 5 54 10 PRT Artificial Sequence Description of ArtificialSequencePeptide 54 Ala Asn Lys Phe Asp Trp Ala Val Glu Thr 1 5 10 55 10PRT Artificial Sequence Description of Artificial SequencePeptide 55 AlaAsn Lys Phe Asp Trp Ala Val Glu Pro 1 5 10 56 10 PRT Artificial SequenceDescription of Artificial SequencePeptide 56 Ala Gly Glu Phe Asp Trp AlaVal Glu Thr 1 5 10 57 10 PRT Artificial Sequence Description ofArtificial SequencePeptide 57 Ala Asp Lys Phe Asp Trp Ala Val Glu Pro 15 10 58 10 PRT Artificial Sequence Description of ArtificialSequencePeptide 58 Ala Asp Arg Phe Asp Trp Ala Val Glu Pro 1 5 10 59 10PRT Artificial Sequence Description of Artificial SequencePeptide 59 SerSer His Phe Gly Trp Ala Val Glu Thr 1 5 10 60 9 PRT Artificial SequenceDescription of Artificial SequencePeptide 60 Leu Ile Cys Phe Val Ile ArgLeu Ala 1 5 61 9 PRT Artificial Sequence Description of ArtificialSequencePeptide 61 Leu Thr Cys Phe Val Ile Arg Phe Ala 1 5 62 9 PRTArtificial Sequence Description of Artificial SequencePeptide 62 Leu IleCys Phe Val Ile Arg Phe Thr 1 5 63 9 PRT Artificial Sequence Descriptionof Artificial SequencePeptide 63 Leu Ala Cys Phe Val Ile Arg Phe Ala 1 564 9 PRT Artificial Sequence Description of Artificial SequencePeptide64 Leu Thr Cys Phe Val Ile Arg Phe Val 1 5 65 9 PRT Artificial SequenceDescription of Artificial SequencePeptide 65 Leu Thr Cys Phe Ile Ile ArgPhe Ala 1 5 66 9 PRT Artificial Sequence Description of ArtificialSequencePeptide 66 Phe Ile Cys Phe Val Ile Arg Phe Ala 1 5 67 9 PRTArtificial Sequence Description of Artificial SequencePeptide 67 Phe ValCys Phe Val Ile Arg Ala Ala 1 5 68 18 PRT Artificial SequenceDescription of Artificial SequencePeptide 68 Leu Gln Leu Ile Tyr Asn LeuThr Leu Cys Glu Leu Asn Gly Thr Asp 1 5 10 15 Trp Leu 69 19 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 69ctgcaagact cgaactgaa 19 70 24 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 70 ggggaattcg ggatagggaa tgtg 24 71 26DNA Artificial Sequence Description of Artificial Sequencesynthetic DNA71 gggggatcct gttggtaata ggtctg 26 72 26 DNA Artificial SequenceDescription of Artificial Sequencesynthetic DNA 72 gggggatcct gttggtaataagtctg 26 73 28 DNA Artificial Sequence Description of ArtificialSequencesynthetic DNA 73 ggtgaattcg ttttatttcc ctccgggc 28 74 18 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 74gatagagtct gcccttag 18 75 18 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 75 ggtttcacct agaatggc 18 76 17 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 76gcttctgaga tgagtga 17 77 18 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 77 caaccaggcg taaacact 18 78 17 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 78ctgagcaatt acagaag 17 79 18 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 79 gactgatggt ctggaaag 18 80 18 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 80ctgtatccga ttcaaacc 18 81 18 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 81 aggttggctg gtggtctt 18 82 18 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 82tcgctcacta cctgtttc 18 83 18 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 83 tgtgcccgcc ttgcctca 18 84 18 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 84aaaccaattg cccccgtc 18 85 18 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 85 tatatcactg tcacagcc 18 86 18 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 86caaattgcca acagaatg 18 87 20 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 87 caacttgacg ctatgtgagc 20 88 20 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 88gccgcggaac catcaagcac 20 89 20 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 89 gactgctagg gcttctgcac 20 90 18 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 90cgttgaccgt agtggagc 18 91 22 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 91 ccccatttcc ctctagcgac tg 22 92 22DNA Artificial Sequence Description of Artificial Sequencesynthetic DNA92 cggccgtgtg gttctcgcca at 22 93 19 DNA Artificial Sequence Descriptionof Artificial Sequencesynthetic DNA 93 gactgcttta cggtctctc 19 94 18 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 94gatgcctgac acattgcc 18 95 19 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 95 ctgcaagact cgaactgaa 19 96 30 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 96gcacggatcc gaattaacat gaaatggggt 30 97 30 DNA Artificial SequenceDescription of Artificial Sequencesynthetic DNA 97 ccacctgcag attcaccgtgagttcgaaag 30 98 30 DNA Artificial Sequence Description of ArtificialSequencesynthetic DNA 98 tgccaggatc cgtgtttaaa tatgttgggg 30 99 30 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 99cgtggaattc atagaaaacg ccaagagcac 30 100 21 DNA Artificial SequenceDescription of Artificial Sequencesynthetic DNA 100 ggggatccagagtttcagcg g 21 101 25 DNA Artificial Sequence Description of ArtificialSequencesynthetic DNA 101 gggaattctg gcacagctga ttgac 25 102 22 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 102ggggatcctt gttaaatatg cc 22 103 18 DNA Artificial Sequence Descriptionof Artificial Sequencesynthetic DNA 103 gggaattcac cacgcatt 18 104 18PRT Porcine reproductive and respiratory syndrome virus 104 105 30 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 105cgtcggatcc tcctacaatg gctaatagct 30 106 30 DNA Artificial SequenceDescription of Artificial Sequencesynthetic DNA 106 cgcgctgcagtgtccctatc gacgtgcggc 30 107 30 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 107 gtatggatcc gcaattggtt tcacctataa 30108 30 DNA Artificial Sequence Description of ArtificialSequencesynthetic DNA 108 ataggaattc aacaagacgg cacgatacac 30

What is claimed as new and desired to be secured by Letters Patent of the United States is:
 1. A composition comprising at least one polypeptide selected from the group consisting of: proteins encoded by one or more open reading frames (ORF's) of an Iowa strain of porcine reproductive and respiratory syndrome virus (PRRSV); proteins with at least 99% but less than 100% amino acid homology with proteins encoded by one or more of ORFs 2 of VR 2385 and VR 2474, ORF 3 of VR 2429, and ORFs 4 of VR 2429 and ISU 1894; proteins with at least 98% but less than 100% amino acid homology with proteins encoded by one or more of ORF 2 of VR 2430 and ORF 5 of VR 2429; proteins with at least 97% but less than 100% amino acid homology with proteins encoded by one or more of ORF 2 of ISU 1894, ORFs 3 of VR 2474 and ISU 1894, ORF 4 of VR 2474 and ORF 5 of ISU 1894; proteins with at least 96% but less than 100% amino acid homology with the protein encoded by ORF 4 of VR 2430; proteins with at least 95% but less than 100% amino acid homology with proteins encoded by one or more of ORF 3 of VR 2430, ORF 4 of VR 2385, and ORF 5 of VR 2474; proteins with at least 94% but less than 100% amino acid homology with the protein encoded by ORF 2 of VR 2431; proteins with at least 93% but less than 100% amino acid homology with the protein encoded by ORF 3 of VR 2431; proteins with at least 92% but less than 100% amino acid homology with the proteins encoded by one or more of ORF 3 of VR 2385 and ORF 5 of VR 2431; proteins with at least 90% but less than 100% amino acid homology with those encoded by one or more of ORFs 5 of VR 2385 and VR 2430; proteins with at least 88% but less than 100% amino acid homology with the protein encoded by ORF 3 of VR 2431; proteins with at least 97% but less than 100% amino acid homology with proteins encoded by one or both of ORF 6 and ORF 7 of an Iowa strain of PRRSV; and combinations thereof.
 2. The composition of claim 1, wherein said polypeptide is encoded by a polynucleic acid sequence selected from the group consisting of the formulas (I), (II), (III) and (IV): 5′-α-β-3′  (I) 5′-α-βγ-3′  (II) 5′β-δ-γ-3′  (III) 5′-α-β-δ-γ-3′  (IV) wherein: β encodes at least one polypeptide, low-virulence mutant thereof, or antigenic or low-virulence fragment thereof encoded by a polynucleotide selected from the group consisting of ORFs 2, 3 and 4 of an Iowa strain of PRRSV; β is at least one copy of an ORF 5 from an Iowa strain of PRRSV or one or more hypervariable regions thereof; γ encodes at least one polypeptide encoded by a polynucleotide selected from the group consisting of ORF 6 and ORF 7 of an Iowa strain of PRRSV; and δ is either a covalent bond or a linking polynucleic acid which does not materially affect transcription and/or translation of the polynucleic acid.
 3. The composition of claim 1, wherein said polypeptide comprises at least one hypervariable region of a protein encoded by an ORF 5 of an Iowa strain of PRRSV.
 4. The composition of claim 1, wherein said polypeptide is selected from the group consisting of proteins with at least 97% amino acid homology with proteins encoded by ORF 6 of VR 2385, VR 2429 (ISU-22), ISU-79 and VR 2431 (ISU-3927); proteins with at least 90% amino acid homology with proteins encoded by ORFs 5 of VR 2385 and VR 2430; proteins with at least 94% amino acid homology with the protein encoded by ORF 2, at least 88% amino acid homology with the protein encoded by ORF 3, and at least 93% amino acid homology with the protein encoded by ORF 4 of VR
 2431. 5. The composition of claim 1, wherein said polypeptide is encoded by a polynucleic acid selected from the group consisting of ORF 2 of VR 2385, ORF 3 of VR 2385, ORF 4 of VR 2385, ORF 5 of VR 2385, ORF 6 of VR 2385, ORF 7 of VR 2385, ORF 2 of VR 2431, ORF 3 of VR 2431, ORF 4 of VR 2431, ORF 5 of VR 2438, ORF 6 of VR 2431, and ORF 7 of VR
 2431. 6. The purified preparation of claim 1, wherein said polypeptide is encoded by a polynucleic acid selected from the group consisting of ORFs 2, 3, 4, 5, 6 and 7 of VR 2385 and VR
 2431. 7. The purified preparation of claim 1, wherein said polypeptide is a homologous protein, and non-homologous residues in said homologous protein are conservatively substituted.
 8. A composition comprising a polypeptide encoded by a polynucleic acid having the formula (V): 5′-ε-ζ-ι-κ-ξ-3′  (V) where: κ is a polynucleotide comprising one or more polynucleotides selected from the group consisting of (a) polynucleotide encoding at least one polypeptide encoded by one or more of ORFs 2, 3 and 4 of an Iowa strain of PRRSV; (b) polynucleotide encoding at least one polypeptide encoded by an ORF 5 from an Iowa strain of PRRSV; (b) polynucleotide encoding at least one polypeptide encoded by an ORF 6 or ORF 7 of an Iowa strain of PRRSV; and (d) operationally linked combinations thereof; and wherein K optionally further comprises a polynucleic acid encoding a conventional marker or a reporter gene; ε which is optionally present, is a 5′-terminal polynucleotide sequence which provides a means for operationally expressing the polynucleotide K; ζ is a polynucleotide of the formula KTVACC, where K is T, G or U, and V is A, G or C; ι is a polynucleotide not more than 130 nucleotides in length; and ξ, which is optionally present and when present may be operationally linked to ε, and is a 3′-terminal polynucleotide sequence which does not suppress the operational expression of the polynucleotide κ.
 9. An antibody to the polypeptide of claim
 1. 10. The antibody of claim 9, wherein said antibody is directed to the protein encoded by ORF
 5. 11. The antibody of claim 10, wherein said antibody is neutralizing.
 12. The antibody of claim 11, wherein said antibody is designated PP5dB4.
 13. The antibody of claim 9, wherein said antibody is directed to the protein encoded by ORF
 4. 14. A method of treating a pig suffering from porcine reproductive and respiratory syndrome, comprising administering an effective amount of the antibody of claim 12 to a pig in need thereof.
 15. A diagnostic kit for assaying a porcine reproductive and respiratory syndrome virus, comprising the antibody of claim 12 and a diagnostic agent which indicates a positive immunological reaction with said antibody.
 16. A method of producing a polypeptide, comprising expressing a polynucleic acid encoding the polypeptide of claim 1 in an operational expression system, and purifying said expressed polypeptide from said expression system. 